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Journal of Bacteriology, October 2000, p. 5841-5848, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evidence of a Role for LytB in the Nonmevalonate
Pathway of Isoprenoid Biosynthesis
Francis X.
Cunningham Jr.,*
Toulouse P.
Lafond, and
Elisabeth
Gantt
Department of Cell Biology and Molecular
Genetics, University of Maryland, College Park, Maryland 20742
Received 13 June 2000/Accepted 26 July 2000
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ABSTRACT |
It is proposed that the lytB gene encodes an enzyme of
the deoxyxylulose-5-phosphate (DOXP) pathway that catalyzes a step at
or subsequent to the point at which the pathway branches to form
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A
mutant of the cyanobacterium Synechocystis strain PCC 6803 with an insertion in the promoter region of lytB grew
slowly and produced greenish-yellow, easily bleached colonies.
Insertions in the coding region of lytB were lethal.
Supplementation of the culture medium with the alcohol analogues of IPP
and DMAPP (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol) completely
alleviated the growth impairment of the mutant. The Synechocystis
lytB gene and a lytB cDNA from the flowering plant
Adonis aestivalis were each found to significantly enhance
accumulation of carotenoids in Escherichia coli
engineered to produce these colored isoprenoid compounds. When combined
with a cDNA encoding deoxyxylulose-5-phosphate synthase
(dxs), the initial enzyme of the DOXP pathway, the
individual salutary effects of lytB and dxs
were multiplied. In contrast, the combination of lytB and a
cDNA encoding IPP isomerase (ipi) was no more
effective in enhancing carotenoid accumulation than ipi
alone, indicating that the ratio of IPP and DMAPP produced via the DOXP
pathway is influenced by LytB.
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INTRODUCTION |
The more than twenty thousand
isoprenoids so far identified (4) include an incredible
variety of essential and secondary compounds, such as carotenoids,
cholesterol, rubber, dolichols, the side chains of quinones, the phytol
tail of chlorophylls, and the prenyl groups of prenylated proteins and
isopentenylated tRNAs. The biosynthesis of all isoprenoids begins with
one or both of the two C5 building blocks of the pathway:
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).
In animals, fungi, and certain bacteria, the synthesis of IPP
occurs via the well-known mevalonate (MVA) pathway that begins with
acetyl coenzyme A (CoA) and proceeds via
hydroxymethylglutaryl-CoA and MVA (16). DMAPP is then
derived from IPP through the action of an IPP isomerase (IPI) enzyme
(35).
In plant chloroplasts, algae, cyanobacteria, and many other
bacteria, an alternative or nonmevalonate pathway, now known
as the deoxyxylulose-5-phosphate (DOXP) pathway, serves
to produce the isoprenoid precursors IPP and DMAPP
(12, 28, 40). This pathway, illustrated for
Escherichia coli in Fig. 1,
utilizes pyruvate and glyceraldehyde-3-phosphate as the initial
precursors rather than acetyl-CoA. The first two steps of the DOXP
pathway, leading to the compound
2-C-methyl-D-erythritol 4-phosphate (MEP), are catalyzed by the enzymes deoxyxylulose-5-phosphate
synthase (DXS) (29, 45) and
deoxyxylulose-5-phosphate reductoisomerase (DXR)
(48). Several subsequent reactions, the order of which is not yet certain, are catalyzed by products of the ygbB,
ygbP, and ychB genes (21, 26, 30, 39).
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FIG. 1.
The DOXP pathway for biosynthesis of the isoprenoid
precursors IPP and DMAPP in E. coli. Gene products
implicated in the pathway are boxed. The order of the reactions
catalyzed by the products of ygbB, ygbP, and
ychB is uncertain. Later reaction steps in the pathway
remain to be determined. Note that a gene encoding IPI, although
present in E. coli, is not routinely found in organisms that
utilize the DOXP pathway (Table 1). The plasmid pAC-LYC
(10), shown to the lower right, encodes enzymes (products of
the crtE, crtB, and crtI genes) that
lead to the synthesis of the pink isoprenoid lycopene from IPP and
DMAPP. Cm, chloramphenicol resistance gene; G-3-P,
D-glyceraldehyde-3-phosphate; GAPD,
glyceraldehyde-3-phosphate dehydrogenase.
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Recent evidence (1, 5, 14, 33, 38) indicates that, in
contrast to the MVA pathway, the DOXP pathway branches and separately
produces DMAPP and IPP. Although E. coli contains an isomerase activity, this activity is low (14, 18), the
ipi gene in this bacterium is dispensable (18),
and ipi is, in any case, not present in the genomes of
many bacteria with the DOXP pathway (Table 1).
Synechocystis strain PCC 6803, for example, lacks a
homologue of ipi (Table 1), and cell extracts of this cyanobacterium and of Synechococcus strain PCC 7942 are
devoid of isomerase activity (14).
The terminal reaction steps in the DOXP pathway have not yet been
established. To identify genes that might encode enzymes that catalyze
these later reactions, we took two different and complementary
approaches. First, the occurrence of the known DOXP pathway genes in 28 completely sequenced bacterial genomes and in yeast, humans, and
the green plant Arabidopsis thaliana was ascertained,
and genes that exhibited the same pattern of occurrence were
identified. Second, we screened bacterial genomic libraries and
plant cDNA libraries for genes or cDNAs that increased the accumulation
of a colored "reporter" isoprenoid compound, lycopene, in
Escherichia coli engineered to produce this pink carotenoid. Common to both the correlative (genomic occurrence) and
functional (lycopene enhancement) screening approaches was the
identification of homologues of the E. coli lytB gene as
prospective DOXP pathway genes. Experimental evidence of a role for
LytB in the DOXP pathway is presented.
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MATERIALS AND METHODS |
Genomic analysis.
The occurrence of homologues of known DOXP
and MVA pathway genes within 28 sequenced bacterial genomes and in
Saccharomyces cerevisiae was determined by using the default
parameters for creation of homologous gene tables at the Microbial
Genome Database for Comparative Analysis (http://mbgd.genome.ad.jp/).
For a few genomes in which the ygbP gene is fused to the
ygbB gene, both genes were considered to be present. Because
results were identical for sequenced genomes of two different strains
of Chlamydia trachomatis, Helicobacter pylori,
and Mycobacterium tuberculosis, each species is listed only
once in the tabulated results (Table 1).
The occurrence of DOXP pathway gene homologues in A. thaliana and in humans was determined by searching the protein,
nucleotide, and expressed sequence tag (EST) databases at GenBank by
using the same significance thresholds as for the microbial database.
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TABLE 1.
Occurrence of known and prospective isoprenoid pathway
genes in members of the Bacteria, Archaea,
and Eucaryota
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Cell strains and culture.
Escherichia coli strains
XL1-Blue MRF' (Stratagene Cloning Systems, La Jolla, Calif.) and Top10
(Invitrogen Corporation, Carlsbad, Calif.) were grown in Luria-Bertani
(LB) medium at 28°C in darkness on a platform shaker at 225 cycles
per min. Cultures grown on solid media (1.5% [wt/vol] Difco Bacto
agar) were incubated at room temperature (ca. 22°C) in darkness. The
growth media were supplemented, as appropriate, with 150 µg of
ampicillin (sodium salt) per ml, 30 µg of chloramphenicol per ml,
and/or 30 µg of kanamycin sulfate per ml.
Cultures of
Synechocystis strain PCC 6803, obtained from Wim
Vermaas (Arizona State University, Tempe) were grown at 30°C
in
liquid BG-11 medium (
36) containing 5 mM TES
[
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic
acid]-KOH (pH 8.2). For agar plates (1.5% [wt/vol] Difco Bacto
agar), 0.3% sodium thiosulfate and 10 mM TES-KOH (pH 8.2) were
included in the medium (
49). The growth media were
supplemented
as needed with kanamycin sulfate, 3-methyl-3-buten-1-ol
(Fluka
66095; Fluka Chemical Corporation, Milwaukee, Wis.), and/or
3-methyl-2-buten-1-ol
(Fluka 66093). Agar plates and liquid cultures
were continuously
illuminated with 20 µE provided by fluorescent
lamps (alternating
tubes of GE cool white and Philips agro), with
cheesecloth used
to reduce the irradiance to the desired
level.
Disruption of the Synechocystis strain PCC 6803 lytB gene.
Genomic DNA was prepared from cells of
Synechocystis strain PCC 6803 as previously described
(49). PCR primers lytB6803N (ATT GCC CCT GGG TAC AAG AC) and
lytB6803C (TCA AGG CAG TGA CCA AGA AAC) were designed to amplify the
lytB gene and flanking DNA of Synechocystis
strain PCC 6803 (23). PCR primers EclytBN (CAC CTT CAA CCT
TGC CGA TAC) and EclytBC (ACC GGC ATT TTC GCA TAA CT) were designed to
amplify the E. coli lytB gene along with the preceding
slpA gene and the two promoters that immediately precede
slpA. PCR was performed with an MJ Research (Waltham, Mass.)
PTC-150-25 MiniCycler with a heated lid and in-sample temperature probe. The Advantage KlenTaq polymerase mix (Clontech Laboratories, Inc., Palo Alto, Calif.) was used with a reaction volume of 50 µl in
100-µl thin-wall tubes. An initial denaturation at 94°C for 1 min
was followed by 10 cycles of 94, 60, and 68°C for 10, 60, and
150 s, respectively, and then 20 to 25 more cycles with an
additional 10 s added to the extension time with each new cycle. The PCR products of the expected size (2.5 kb for the
Synechocystis lytB) (Fig. 2)
were purified by electrophoresis in a 1% (wt/vol) agarose gel,
recovered by using the Geneclean kit (Bio 101, Inc., Carlsbad, Calif.)
and cloned in the plasmid vector pSTBlue1 (Novagen, Inc., Madison,
Wis.). A 2.2-kb fragment containing Synechocystis lytB was
excised with the BamHI and SmaI sites contained
within the genomic PCR product and inserted into the
BamHI and Klenow-blunted HindIII sites of
pTrcHisA (Invitrogen) and in the BamHI and EcoRV sites of pBluescript SK
(Stratagene) to yield p6803lytBTrcA and p6803lytBSK. Digestion of p6803lytBTrcA with BamHI and
HindIII, filling in of the ends with the Klenow fragment
of DNA polymerase, and ligation to recircularize yielded plasmid
p6803lytBfus, in which the lytB of Synechocystis
was fused in frame to a small peptide under the control of the strong
Trc promoter.
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FIG. 2.
Schematic illustration of Synechocystis
strain PCC 6803 genomic DNA encompassing the lytB
gene. A kanamycin resistance gene (Kanr) was inserted into
the NheI site immediately upstream of the lytB
gene.
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The kanamycin resistance gene from Tn
903 was excised
from plasmid pBR329K (
3) with
PstI, the
ends were made blunt with
mung bean nuclease, and the Kan
r
gene was inserted into the blunted
HindIII site near the
beginning
of the
lytB gene (Fig.
2) in p6803lytBTrcA, into
the blunted
EcoRI
site in the
lytB gene in
p6803lytBSK, and into the blunted
NheI
site upstream of
lytB in p6803lytBSK. After transformation of
E. coli and selection of the appropriate transformants, plasmid
minipreps were prepared (
11). The plasmids were linearized
by
digestion with
BamHI, the reaction mixtures were
extracted twice
with phenol and then once with CHCl
3, and
the DNA was precipitated
with sodium acetate and ethanol
(
42), washed with ice-cold 70%
ethanol, and resuspended in
sterile Tris-EDTA buffer. Transformation
of
Synechocystis
strain PCC 6803 was performed as described by
Williams (
49),
except that the concentrated cultures were grown
for 24 h after
addition of the transforming DNA and spread directly
on BG-11 agar
plates supplemented with 10 µg of kanamycin sulfate
per ml. Colonies
appearing on these plates were streaked onto
plates with 25 µM
kanamycin, and colonies arising on these plates
were streaked, in turn,
on plates containing 50 µM
kanamycin.
Library construction and screening.
A unidirectional cDNA
library in lambda ZAPII, using mRNA isolated from immature and
developing flower buds of Adonis aestivalis (pheasant's
eye), was constructed (by Stratagene) with EcoRI and XhoI adapters. The library was excised en masse to produce a
phagemid library (according to the manufacturer's instructions), which was then introduced into E. coli strain XL1-Blue MRF' that
had been engineered to accumulate the pink carotenoid lycopene
(10). Transfected cultures were spread on LB agar plates to
yield a density of ca. 2,000 to 10,000 colonies per large petri plate (150 mm in diameter and containing ca. 100 ml of growth medium). Both
liquid and solid growth media contained ampicillin to select and
maintain transformants and chloramphenicol to maintain the plasmid
required for lycopene production (pAC-LYC; Fig. 1) (10). Plates were incubated at room temperature in darkness and examined visually after 2 days and daily thereafter for 7 to 10 days. The rare
dark pink colonies observed in the midst of the multitude of paler pink
colonies were selected, the library plasmids within the cells of these
colonies were recovered, and the sequences of the cDNA inserts were
determined. Full details of the screening protocol are given elsewhere
(9, 10).
A related approach to identification of prospective isoprenoid pathway
genes has been described by Hemmi et al. (
20), who
mapped
mutations that reduced lycopene accumulation in
E. coli to
29 loci. Although
ipi was found in this screen,
lytB,
gcpE,
and
dxs were not among the
prospective isoprenoid pathway genes
identified this
way.
Plasmids and plasmid constructs.
A 0.85-kb
KpnI-EcoRI fragment containing most of the
Arabidopsis ipi cDNA (8) was excised from the
original library plasmid and ligated in the corresponding sites of
plasmid pTrcHisB (Invitrogen). Subsequent digestion with
NcoI and XhoI, filling-in with the Klenow fragment of DNA polymerase, and ligation to recircularize yielded a
plasmid in which the coding region of ipi, lacking the first 48 codons, is fused in frame to the strong bacterial Trc
promoter and 6 additional codons that specify the amino acids MSRSAA.
An EcoRV-KpnI fragment, containing the cDNA and
fused Trc promoter, was excised and inserted in the
corresponding sites of pBluescript SK to produce the plasmid pAtipiTrc.
A marigold
dxs cDNA in the original cloning vector
(pBluescript SK
), here referred to as pTedxs, has already been
described
(C. P. Moehs, L. Tian, and D. DellaPenna, submitted for
publication).
An
Adonis lytB cDNA (GenBank accession no.
AF270978), along
with the
lacZ promoter immediately upstream
of it in the cloning
vector (pBluescript SK
), was excised from the
original library
clone (pAplytB) with
PvuII and inserted
into the Klenow-blunted
SalI site of plasmid pTedxs to yield
pdxs/lytB.
An
EcoRV-
EcoRI fragment containing the
Arabidopsis ipi1-
Trc promoter fusion (see above)
was excised from pAtipiTrc, treated
with the Klenow enzyme to blunt the
ends, and inserted into the
blunted
SalI site downstream of
the marigold
dxs cDNA in pTedxs,
in the blunted
KpnI site downstream of the
Adonis lytB cDNA in
pAplytB, and in the blunted
XhoI site of pdxs/lytB. The
resulting
plasmids are referred to as pdxs/ipiTrc, plytB/ipiTrc, and
pdxs/lytB/ipiTrc.
Assay of carotenoid accumulation.
Competent cells of
lycopene-, 
-carotene-, and zeaxanthin-accumulating E. coli strain Top10 (9, 10, 47) were prepared (6) from cultures grown at 28°C in darkness with shaking.
After transformation with various plasmids, cells were spread on agar plates with the appropriate selective agents and incubated at room
temperature for 2 to 3 days in darkness. Culture tubes containing 5 ml
of LB medium (with ampicillin and chloramphenicol) were each inoculated
with a single fresh colony, and the cultures were grown for 48 h
at 28°C with shaking, conditions previously reported as optimal for
carotenoid accumulation in E. coli (41). Two 1.5-ml aliquots from each tube were harvested by centrifugation in
microcentrifuge tubes (90 s at maximum speed in an Eppendorf 5415C
microcentrifuge). The well-drained pellets were extracted with 1.5 ml
of chloroform-methanol (2/1 [vol/vol]) for 3 h in darkness, with
occasional gentle mixing by inversion of the tubes. Extracts were
clarified by centrifugation for 10 min at maximum speed in the
microcentrifuge. The A480.5 (the major peak in
the visible region of the absorption spectrum of lycopene) or the A458.5 (the major peak for -carotene and
zeaxanthin) was determined with an accumulation time of 4 s and
the slit width set to 4 nm in a Perkin-Elmer lambda 6 spectrophotometer. A650 was also monitored to
ensure that the reading at 480.5 (or 458.5) nm was not due to turbidity
of the extract. At least 6 (zeaxanthin and -carotene) or 12 (lycopene) individual transformants were analyzed for each plasmid construct.
Nucleotide sequence accession number.
The Adonis
aestivalis lytB cDNA has been assigned GenBank accession no.
AF270978.
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RESULTS |
The genomic occurrence of known DOXP pathway genes is
matched only by lytB and gcpE.
The occurrence of
homologues of the known DOXP pathway genes dxs,
dxr, ygbB, ygbP, and ychB
was determined for 22 bacterial (including the cyanobacterium
Synechocystis strain PCC 6803) and 6 archaebacterial genomes
for which the complete sequences were available in public databases.
Their occurrence in the yeast genome, in the substantially completed
Arabidopsis thaliana genome and EST databases, and in the
human genome and EST databases was also ascertained. For comparison,
the occurrence of four MVA pathway genes and ipi was
determined as well. The results of this analysis are displayed in Table
1.
DOXP pathway genes
dxs,
dxr, and
ygbB
were present in all but five of the bacteria, in none of the six
archaea, and in
Arabidopsis,
but not in yeast or humans. The
ygbP and
ychB gene occurrence
patterns were
similar, but not quite identical to that of these
first three. The
exact occurrence pattern exhibited by
dxs,
dxr,
and
ychB was displayed by only two other genes, both of
unknown
or uncertain function:
lytB and
gcpE. An
observed co-occurrence
of genes in this type of analysis has been found
to be a strong
predictor of a functional linkage (
31,
34).
Therefore,
lytB and
gcpE must be considered
strong candidates for the few DOXP
pathway genes that remain to be
identified.
Alcohol analogues of IPP and DMAPP complement an insertion
immediately upstream of Synechocystis lytB.
The
lytB gene of the cyanobacterium Synechocystis
strain PCC 6803 was obtained by PCR amplification of genomic
DNA. A kanamycin resistance gene from Tn903 was inserted
into either the EcoRI or HindIII site in the
coding region of lytB or the NheI site immediately upstream of the coding region (Fig. 2), and
Synechocystis was separately transformed with linearized DNA
containing these constructs. Colonies appearing after transformation
with constructs containing the Kanr gene in the
HindIII or EcoRI sites of lytB
were quite small, nearly transparent in appearance, and did not survive
restreaking on 25 µM kanamycin plates. Serial restreaking on agar
plates containing increasing concentrations of kanamycin (see Materials
and Methods) did, however, yield a
lytB::Kanr strain with the insertion
into the NheI site of all copies of the genome (Fig.
3). Colonies produced by this strain grew
slowly on the 50 µM kanamycin plates, were greenish-yellow in color
(Fig. 4, upper panel), and eventually
became bleached despite a relatively low light regimen (20 µE of
continuous white light).
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FIG. 3.
PCR amplification of lytB in
Synechocystis PCC6803 using genomic DNA obtained
from the wild-type strain and from a mutant with a Kanr
insertion in the NheI site immediately upstream of
lytB. The ca. 3.7-kb PCR product from the insertion mutant
lytB(NheI)::Kanr, displayed
in the right lane of this 1% agarose gel, is the expected 1.2 kb
larger than the wild-type product of about 2.5 kb (middle lane). No
trace of the wild-type (i.e., uninterrupted) gene is discernable in the
mutant lane. The PCR primers were those used for the initial
amplification of lytB and flanking regions (see Fig. 2 and
Materials and Methods) and lie outside the BamHI and
SmaI sites that define the cloned DNA fragment used to
transform Synechocystis. The standards lane (left lane)
contains DNA fragments of the indicated sizes.
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FIG. 4.
Impaired growth of Synechocystis strain PCC
6803 lytB(NheI)::Kanr
mutant strain on BG-11 agar plates containing 50 µg of kanamycin per
ml (upper panel) is much improved by supplementation of the growth
medium with 2.5 mM (each) 3-methyl-3-buten-1-ol and
3-methyl-2-buten-1-ol (lower panel). The dark blue-green color of the
colonies in the lower panel is comparable to that displayed by the wild
type grown on BG-11 agar plates lacking kanamycin and alcohols.
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It was reported earlier (
25,
48) that the alcohol analogue
(2-
C-methylerythritol [ME]) of the DOXP pathway
intermediate
MEP could chemically complement a mutation in an
E. coli gene
(
yaeM [now known as
dxr])
encoding an enzyme (DXR) (Fig.
1) that
catalyzes the formation of this
compound. We reasoned that the
alcohol analogues of IPP and DMAPP
(3-methyl-3-buten-1-ol and
3-methyl-2-buten-1-ol, respectively) might
similarly be taken
up by
Synechocystis, phosphorylated by
endogenous kinases, and
thereby serve to chemically complement
mutations that eliminate
or reduce the production of IPP and/or DMAPP
via the DOXP pathway.
Supplementation of solid media with
3-methyl-3-buten-1-ol and
3-methyl-2-buten-1-ol completely
ameliorated the impairment in
growth observed for the
lytB(
NheI)::Kan
r insertion
mutant of
Synechocystis. Colonies formed by cells of
this mutant on agar plates containing the alcohols
exhibited the
dark blue-green color (Fig.
4, lower panel) typical of
colonies
formed by wild-type
cells.
Cyanobacterial and plant lytB enhance isoprenoid
accumulation in Escherichia coli.
The
Synechocystis lytB gene, fused to the strong bacterial
Trc promoter in the plasmid vector pTrcHis (see
Materials and Methods), was introduced into a strain of
Escherichia coli engineered to produce the pink-colored
isoprenoid compound lycopene (Fig. 1). Cells of E. coli
containing this plasmid form colonies much darker pink than those
formed by cells containing the empty plasmid vector (Fig.
5). Much the same enhancement of lycopene
accumulation has previously been achieved by increasing the copy number
of two genes that encode enzymes of the isoprenoid pathway in E. coli: dxs (19, 32) and ipi
(7, 14, 22, 46, 47).
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FIG. 5.
A lycopene-accumulating strain of E. coli
that contains a plasmid with an Adonis aestivalis cDNA
encoding LytB (pAplytB; see Materials and Methods) yields colonies much
darker (the dark pink colonies) and containing much more of this pink
isoprenoid than colonies produced by cells in which the second plasmid
is the empty cloning vector (pale pink colonies).
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A flower cDNA library of the green plant
Adonis aestivalis
(pheasant's eye) was screened in the lycopene-accumulating
E. coli strain for clones that produced a dark pink
colony color. Two
of the selected dark pink colonies contained plasmids
with cDNAs
encoding homologues of the
Synechocystis LytB
(most of the cDNAs
were of
ipi) (
8). Although
differing in length, the two Adonis
lytB cDNAs were
otherwise identical in sequence, and both were
in the correct frame to
produce a fusion protein (with an additional
41 or 50 amino acids
derived from the presumed 5' untranslated
region of the cDNA, from
the N-terminal adapter used to produce
the cDNA, and from
lacZ) under the control of the
lacZ promoter
of
the cloning vector (pBluescript SK
). Reading from the first
methionine, the 464-amino-acid polypeptide predicted by the
Adonis
cDNAs has an N-terminal extension of 54 amino acids when aligned
with the closely related (more than 60% identity)
Synechocystis LytB (Fig.
6).
The
Adonis N-terminal extension has the
characteristics
expected of a plastid targeting sequence, and the
polypeptide
was indeed predicted to be targeted to the chloroplast
by the
program ChloroP (
13;
http://www.cbs.dtu.dk /services/ChloroP/).
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FIG. 6.
Alignment of amino acid sequences deduced from a
bacterial (Escherichia coli; Ec), a cyanobacterial
(Synechocystis strain PCC 6803; Sy), and a plant
(Adonis aestivalis; Aa) lytB gene or cDNA.
Residues identical in a given position for the three sequences are in
white text on a black background. Where two of the three are identical,
residues are in black text on a grey background. A pound sign (#) below
the alignment denotes a position at which the amino acid residues are
identical in more than 90% and/or similar in 100% of 22 sequences
deduced from lytB homologues for which the complete coding
regions are currently available in GenBank (5 April 2000). GenBank
accession numbers are as follows: A. aestivalis,
AF270978:27-1421; E. coli, AE000113:5618-6568; and
Synechocystis strain PCC 6803, D64000:46364-47584.
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The amounts of lycopene produced by liquid cultures of
E. coli containing plant cDNAs encoding LytB
(
Adonis), IPI (
Arabidopsis),
and DXS (marigold)
were quantified and compared to a control strain
containing the empty
cloning vector (Table
2).
E. coli strains
producing the carotenoids zeaxanthin (Table
2) and
-carotene
(data not shown; results were essentially the same as for
the
lycopene and zeaxanthin strains) were also employed. The
lytB cDNA (the longer of the two selected clones; see above)
increased
lycopene,
-carotene, and zeaxanthin accumulation by about
1.5-fold.
The
Synechocystis lytB gene also yielded a
1.5-fold increase (when
fused to the strong
Trc promoter;
data not shown). The plant cDNAs
encoding IPI and DXS were a bit more
effective, producing a slightly
more than twofold increase in
carotenoid accumulation.
Combinations of
lytB and
dxs or
ipi
and
dxs were substantially and
significantly more effective than any individual cDNA in
enhancement of
carotenoid pigment accumulation (Table
2). Because
the increase
obtained with the combination of the two cDNAs fall
within the range of
values obtained by multiplying the individual
effects, the influence of
the
Adonis lytB cDNA on carotenoid accumulation
can be said
to be independent of that provided by the marigold
dxs cDNA.
The individual effects of
ipi and
dxs were also
multiplicative,
or nearly so, in combination. With these results to
establish
that sink or substrate supply limitations are not
significant,
it is noteworthy that the combination of
ipi and
lytB yielded
no more carotenoid pigment
than
ipi alone, whether the cDNAs were
combined in a single
plasmid (Table
2) or introduced on separate,
compatible plasmids
that individually bestowed the expected enhancement
(data not
shown).
lytB mRNA is elevated in plant cells specialized for
isoprenoid biosynthesis.
mRNAs encoding enzymes of the isoprenoid
pathways have been shown to be well represented in a cDNA library
constructed with mRNA obtained from certain isoprenoid-rich cells
(glandular trichomes) of Mentha piperita (mint)
(27). Although not well represented in the plant EST
database in general, we found the frequency of cDNAs encoding an LytB
homologue to be extraordinarily high in a collection of ESTs
obtained from this mint glandular trichome library: 11 of the
1,316 ESTs deposited as of 5 April 2000 (accession no. AW254730,
AW254735, AW254890, AW254988, AW255092, AW2551238, AW255223, AW255312,
AW255412, AW255711, and AW255721). Almost the entire M. piperita
lytB cDNA sequence can be assembled from these EST sequences.
Coincidentally, the frequency of dxs cDNAs in the deposited
mint ESTs is exactly the same: 11 of 1,316 (0.84%).
| |
DISCUSSION |
The case for lytB.
The phenotype of the
Synechocystis
lytB(NheI)::Kanr mutant (Fig. 4)
and the lethality of insertions in the coding region of this gene are
in accord with a role for lytB in isoprenoid biosynthesis. Because chlorophyll is one of the major isoprenoids produced in Synechocystis and because the inorganic growth medium that
was used required photosynthesis for growth of the organism, an
impairment of the DOXP pathway would be expected to result in a pale
green and easily bleached colony color with a diminished rate of
growth. Although we attribute the observed phenotype of the
lytB(NheI)::Kanr mutant to
reduced transcription of the lytB gene, we do not have direct evidence of this. The Kanr in this insertion mutant
resides in what is also the putative promoter region of an open reading
frame (s110319) that precedes lytB and reads in the
opposite direction (Fig. 2). Our data do not rule out an
influence on this hypothetical gene. However, the deduced product of
this gene has no obvious homologues in the protein databases, and
therefore it is unlikely to be either an isoprenoid pathway gene or
essential to the organism, if indeed it is transcribed. In combination
with the genomic occurrence pattern of lytB relative
to DOXP pathway genes (Table 1), the significant salutary effect of the
Adonis and Synechocystis lytBs on carotenoid
production in E. coli (Fig. 5 and Table 2), and the high
frequency of lytB in a cDNA library of isoprenoid-rich mint
glandular trichomes (see Results), the chemical complementation of the
pale green, easily bleached Synechocystis
lytB(NheI)::Kanr mutant with
alcohol analogues of IPP and DMAPP (Fig. 4) makes a compelling, albeit
not unequivocal, case for the involvement of lytB in the
DOXP pathway.
The choice of
Synechocystis as the experimental organism for
insertional inactivation of
lytB appears to have been a
fortuitous
one. It does not appear that
E. coli is similarly
able to utilize
the alcohol analogues of IPP and DMAPP for isoprenoid
biosynthesis.
Supplementation of the culture media with these
compounds did
not increase carotenoid accumulation in liquid
cultures of
E. coli, nor yield more deeply pigmented
colonies on agar plates
(data not
shown).
What is otherwise known of lytB?
Relatively little
is known of lytB or of the polypeptide encoded by this gene.
(Note that a Streptococcus pneumoniae murein hydrolase, also
referred to as LytB [15], is unrelated to the lytB gene under discussion here.) The amino acid sequences
predicted by the E. coli, Synechocystis, and
Adonis lytB genes provide no obvious indication (e.g.,
informative sequence motifs or significant resemblance to
polypeptides of known function) as to the function of the
polypeptide. Certain temperature-sensitive mutations in the
E. coli lytB gene have been reported to confer a
tolerance to penicillin (17), a phenotype thought to derive
from an influence of lytB on the stringent response
(37). A tolerance of polymyxin B was also attributed to
mutations in lytB in Burkholderia pseudomallei (2). These observations are not inconsistent with a role for lytB in the DOXP pathway. Any inhibition of the biosynthesis
of the two major classes of isoprenoids produced in E. coli,
dolichols and respiratory quinones (43), likely would impact
cell wall biosynthesis (dolichols) (24) and polymyxin B
uptake (quinones). The importance of the DOXP pathway to wall synthesis
is made clear by the most visible response of E. coli to
inhibition of the DOXP pathway by the chemical inhibitor fosmidomycin:
the formation of spheroplasts (44).
What might be the function of LytB in the DOXP pathway?
It has
become apparent that the DOXP pathway leads separately to DMAPP and IPP
(1, 5, 14, 33, 38) (see the introduction and Fig. 1). What
remains uncertain is whether distinct enzymes lead to each of these
C5 isomers or, perhaps more conservatively, whether a
single enzyme of the pathway yields both compounds or immediate
precursors thereof. The nonadditivity of the effects of ipi
and lytB on carotenoid accumulation in E. coli
(Table 2) indicates that the product of lytB somehow
influences the ratio of DMAPP and IPP formed by means of the DOXP
pathway in E. coli, although LytB is not itself an isomerase
(14, 38). It would seem, therefore, that LytB likely acts at
or subsequent to the branch point of the DOXP pathway. Experiments to
ascertain the biochemical function of LytB are in progress.
| |
ACKNOWLEDGMENTS |
This work was supported primarily by grants from DOE
(DEFG0298ER2032) and NSF (MCB 9631257) with additional support provided by Monsanto, Quest International, and Zeneca Plc.
We are grateful to Charles P. Moehs and Dean DellaPenna of the
University of Nevada at Reno for providing the marigold dxs cDNA and to Yuri Ershov and Raymond Gantt for critical reading of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Cell Biology and Molecular Genetics, Microbiology Building, Campus Dr., University of Maryland, College Park, MD 20742. Phone: (301) 405-1035. Fax: (301) 314-9489. E-mail: fc18{at}umail.umd.edu.
| |
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Abstract
Introduction
