From the Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan
Received for publication, March 14, 2003
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ABSTRACT |
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INTRODUCTION |
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IleRS, the target of antibiotic pseudomonic acid, is one of the aminoacyl-tRNA synthetases (aaRSs), which play a crucial role in the first step of protein biosynthesis, because they are responsible for accurate charging of specific tRNAs with their cognate amino acids (10, 11). Twenty amino acid-specific aaRSs can be divided into two classes, each of which contains 10 enzymes (11, 13, 14). Class I aaRSs, including IleRS, each display two short common consensus sequences, called HIGH and KMSKS, which form a Rossman nucleotide binding fold and play an important role in enzyme catalysis. Furthermore, based on their species-specific conserved sequences, aaRSs can generally be subdivided into either a eubacteria type or an archaeal/eukaryote type (12).
Like other general antibiotic-producing organisms, P. fluorescens is entirely insensitive to the antibiotic it produces, pseudomonic acid (18). Antibiotic-producing bacteria have several common strategies to prevent their own antibiotics from killing them (19). By kinetic analysis it has been revealed that the affinity of P. fluorescens crude IleRS for pseudomonic acid is dramatically lower than that of Escherichia coli (2, 18). It has thus been considered that the difference in the molecular structure of IleRS causes difference in affinity for the antibiotic (the difference in inhibition) and that this structural alteration makes P. fluorescens cells highly resistant to pseudomonic acid.
In this report we provide evidence for two distinct IleRSs (IleRS-R1 and IleRS-R2) in P. fluorescens. The former is an enzyme that we reported earlier (25), and the latter is a quite newly found enzyme that has a markedly reduced affinity for the antibiotic and thereby confers upon the organism a strong resistance to pseudomonic acid. We concluded that the self-defense of the pseudomonic acid producer might be caused mainly by the production of an additional IleRS. The amino acid sequence deduced from the novel ileS gene was similar to eukaryotic IleRSs and evolutionarily distinct from IleRS-R1. The physiological significance of two ileS genes in pseudomonic acid-producing P. fluorescens, and the origin of the newfound ileS gene is also discussed.
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EXPERIMENTAL PROCEDURES |
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Culture Media and Growth ConditionsE. coli cells were grown in LB or SOB medium (45). P. fluorescens cells were grown at 26 °C in either LB culture medium or in agar medium (pH 7.0), the latter containing 10 g of Bacto-tryptone, 10 g of Bacto-proteose peptone number 3, 1.5 g of dipotassium phosphate, and 1.5 g of magnesium sulfate (per liter).
Enzyme Assay of IleRS and Kinetic StudiesAminoacylation activities were measured in the standard mixture (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 40 mM KCl, 50 µM L-[U-14C]isoleucine (50 µCi/mmol), 1 or 4 mM ATP, 5 mg/ml E. coli tRNA, and appropriate amounts of IleRS). Kinetic constants were determined as described previously (25).
Preparation of Crude Cell ExtractP. fluorescens cells were cultured in LB medium (10 ml) at 26 °C for 15 h. The cells were harvested by centrifugation at 12,000 x g, and the pellet was suspended in buffer A5 (20 mM potassium phosphate (pH 7.0), 10% glycerol, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride). The suspensions were sonicated, and the cell debris was pelleted by centrifugation at 10,000 x g for 15 min. The supernatant was further ultracentrifuged at 165,000 x g for 2 h and then dialyzed overnight against buffer A5. The crude enzyme was used for kinetic analysis. The protein concentrations were determined by the method of Lowry (see Ref. 46).
Activity Separation of the Two IleRS of P. fluorescens by DEAE-Sephacel ChromatographyP. fluorescens NCIB10586 cells were cultured in LB medium (1 liter) at 26 °C for 12 h. Cells were harvested by centrifugation at 10,000 x g, and the pellet was suspended in buffer A5. The suspension was sonicated, and the cell debris was pelleted by centrifugation at 10,000 x g for 10 min. Subsequently, the supernatant was ultracentrifuged at 100,000 x g for 6 h. The solution was applied directly to a DEAE-Sephacel column (Amersham Biosciences) equilibrated with the buffer containing 20 mM potassium phosphate (pH 7.0), 0.5 mM DTT, 10% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride (buffer A1). After the column was washed with buffer A1, proteins were eluted with a linear gradient of 0 to 0.4 M KCl, followed by buffer A1 containing 0.4 M KCl, and then by buffer A1 containing 1 M KCl. The aminoacylation activity of the fractions was measured at 30 °C for 20 min.
Purification of IleRS-R2 from P. fluorescens NCIB10586 All steps described were performed at 4 °C. Frozen P. fluorescens cells (110 g) were suspended in buffer A5 and sonicated with an ultrasonicator. The crude cell extract was centrifuged at 30,000 x g for 20 min. The precipitate was resuspended in buffer A5, ultrasonicated, and ultracentrifuged at 100,000 x g for 6 h. After the resulting supernatant was dialyzed for 24 h against buffer A1, the solution was applied to a column of DEAE-Sephacel equilibrated with buffer A1. The column was washed, developed with 0 M to 0.4 M KCl gradient, and followed by buffer A1 containing 1 M KCl. Active fractions of eluate were collected. The DEAE-Sephacel fraction was concentrated by ultrafiltration and dialyzed against buffer A1, and then (NH)2SO4 was added to the final concentration of 1 M. This solution was loaded on a column of n-butyl-Toyopearl (Tosoh) equilibrated with buffer same as buffer A1 but containing 1 M (NH)2SO4. After the column was washed with buffer A1 containing 1 M (NH)2SO4, proteins were eluted with a linear gradient of 1 to 0 M (NH)2SO4. Active fractions of eluate were collected, concentrated by ultrafiltration, and dialyzed against buffer A1. An n-butyl-Toyopearl fraction was applied to a column of DEAE-Toyopearl (Tosoh) equilibrated with buffer A1. The column was washed, and then proteins were eluted by a linear gradient of 0 to 0.35 M KCl. Active fractions were pooled and dialyzed against the buffer containing 20 mM potassium phosphate (pH 7.0), 0.5 mM DTT, and 20% glycerol (buffer B). The dialyzed solution was loaded onto a column of hydroxyapatite (Koken Biochemicals) equilibrated with buffer B. The column was washed and then proteins were eluted with a linear gradient of 0.02 to 0.35 M potassium phosphate. Active fractions were pooled, dialyzed against buffer B, and then concentrated. The homogeneity of the enzyme preparation was evaluated by a second hydroxyapatite chromatography. The concentrated solution was applied to the column equilibrated with buffer B. After the column was washed, IleRS-R2 was eluted with a gradient of 0.02 to 0.2 M potassium phosphate. Proteins were detected by absorbance at 280 nm. The eluted IleRS-R2 protein was dialyzed against the buffer containing 20 mM potassium phosphate (pH 7.0), 2 mM DTT, and 50% glycerol. It was then stored at 20 °C (1.14 mg). To analyze the N-terminal amino acid sequence, purified IleRS-R2 was subject to 10% SDS-polyacrylamide gel and transferred to Immobilon P membrane (Millipore). Amino acid sequences were determined as described previously (25).
PCR Amplification and Identification of P. fluorescens ileS2
Gene For the design of degenerate oligonucleotide primers, we used
highly conserved regions near the KMSKS region among known IleRSs, but we did
not use regions shared with other class I aaRSs. An upstream region set of two
primers was designed from amino acid sequences "DVWF(E/D)SG"
(5'-GAYGTNTGGTTYGANTSNGG-3' named V1 (where Y is C + T, S is G +
C, and N is A + G + C + T), and "DCWFESG"
(5'-GAYTGYTGGTTYGAYTSNGG-3' named C1). Downstream set of the two
primers were from "QTRGWFY" (5'-ARAACCANCCNCKNGTRTG-3'
named T1; K is G + T) and from "DQ(H/Y)RGWF"
(5'-AACCANCCNCKRTRYTGRTC-3' named YH1; R is A + G)
(Fig. 5, red arrows
above the sequence alignment). These four primers allow for all codon
variations. PCR was carried out using P. fluorescens NCIB10586
genomic DNA as a template. An amplified DNA fragment of 130 bp was
extracted from agarose gel, blunt-ended, cloned into the HincII site
of pUC19, and introduced into E. coli DH5
. The DNA inserted
from the isolated plasmid (pNO47) was sequenced. pNO47 was used as a PCR
template to prepare the probe for Southern blotting.
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DNA Probes133-bp DNA fragment, amplified using C1 and T1
primers, was gel-purified and radiolabeled with [-32P]dCTP
by a random primer labeling kit (Takara Shuzo). The radiolabeled probe was
used directly for Southern blot analysis and screening of the library. The
ileS1 probe (a 126-bp DNA fragment amplified from
ileS1-containing plasmid using V1 and YH1 primers) was also
radiolabeled and used for Southern blot analysis.
Cloning and Sequencing of P. fluorescens ileS2 GeneFor
cloning, EcoRI-digested P. fluorescens DNA was fractionated
by agarose gel electrophoresis. An 8.5-kbp fraction of DNA fragments
identified by Southern blot analysis was electroeluted from the gel, ligated
into EcoRI-treated pTWV228, and introduced into E. coli
DH5
cells. E. coli cells transformed with plasmids were
subjected to colony hybridization analysis using the radiolabeled 133-bp
probe, which gave two strong hybridization signals out of 3000 recombinants.
An isolated clone (pR2), carrying the ileS2 insert on an 8.3-kbp DNA
fragment, a structural gene and
1000 bp of the flanking region, was
completely sequenced.
Western Blot AnalysisEach portion of the DEAE-Sephacel column fraction of P. fluorescens cell extract was fractionated by electrophoresis through an SDS-10% polyacrylamide gel and transferred onto an Immobilon membrane. The protein of interest was detected using specific antibodies and visualized by the ECL system (Amersham Biosciences).
Plasmid Construction and Expression of IleRSs in E. coliThe
construction of plasmid pEXR1 (the former name is pPFNB7) was described
previously (25). To construct
pEXR2, the P. fluorescens ileS2 gene with PCR-modified 5'- and
3'-ends was ligated into the NdeI/HindIII
double-digested pEXPCR expression vector. The primers used were
5'-GACAGGTGTGACAT(ATG)AGTACGGAAGGAAGTGGGCC-3' and
5'-ACCAGTCGGCAAGCTT(TCA)GGCCAGTACGCTACGGCGC-3' (the
restriction enzyme sites are underlined, and the initiation codons or stop
codons on the reverse strands are in parentheses). Plasmid pR2 was used as a
template. The construct was introduced into DH5, and the ileS2
gene was expressed as described previously
(25).
Overexpression and Purification of Recombinant IleRS
Proteins Recombinant IleRSs were overexpressed and purified as
follows. To prepare IleRS-R1 and IleRS-R2, pEXR1 and pEXR2 were used,
respectively, to transform E. coli strain Ts331. Transformants were
grown in 3 liters of SOB medium containing ampicillin (50 µg/ml) and
isopropyl-1-thio--D-galactopyranoside (0.5 mM) at
30 °C for 10 h with shaking. Cells were harvested and suspended in buffer
A1 containing 2 µg/ml antipine, 2 µg/ml leupeptin, 1 µg/ml pepstatin,
and 1 µg/ml chymostatin. The cells were broken by sonication, and crude
extracts were centrifuged at 30,000 x g for 20 min. The
supernatants were subjected to ultracentrifugation at 100,000 x
g for 6 h to remove cell debris and ribosomes. Purified IleRS
proteins were obtained by three steps of column chromatography (DEAE-Toyopearl
(or DEAE-Sephacel), n-butyl-Toyopearl, and hydroxyapatite) as in the
case of the purification of native IleRS-R2 from P. fluorescens.
Active fractions were pooled, concentrated, and dialyzed against buffer B.
Purified IleRS-R1 and IleRS-R2 were used for preparing polyclonal antibodies.
The enzyme activities of recombinant IleRS-R1 and IleRS-R2 expressed in E.
coli cells were clearly separated from endogenous E. coli IleRS
activity by column chromatography, and little or no endogenous IleRS activity
can remain in these purified recombinant P. fluorescens IleRSs.
Complementation AnalysisE. coli temperature-sensitive ileS mutant Ts331 was used for complementation analysis. Ts331 transformed with ileS-containing plasmids were grown at 42 °C on LB agar plates.
In Vivo Pseudomonic Acid ResistancePseudomonic acid was diluted by 0.8% LB agar media (100 µl) at 10, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 600, 800, and 1000 µg/ml in microtiter plates. Test organisms were grown at 30 °C overnight in the LB medium. The overnight cultures were diluted to a concentration of 0.5 x 109 cells/ml by the LB medium, and then 2-µl spots (106 cells) were inoculated onto the surfaces of the microtiter plates. The plates were incubated at 30 °C for 24 h. Pseudomonic acid sensitivity was determined as the lowest concentration needed to inhibit cell growth completely.
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RESULTS |
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Evidence for Two IleRSs Conferring Distinct Pseudomonic Acid ResistanceThis hypothesis was proved by DEAE-Sephacel column chromatography, in which two peaks of IleRS activity were detected (Fig. 2). Western blot analysis using anti-IleRS-R1 antibody showed that the IleRS-R1 was fractionated in the first peak eluting at a lower salt concentration (0.25 M KCl) but not in the second peak (Fig. 3). With the addition of 20 µM pseudomonic acid to IleRS-R1, the peak seen earlier was completely abolished, whereas the second peak (the 0.4 M KCl fraction) was fully active under the same conditions (Fig. 2), suggesting that this fraction contains a newly found IleRS that is highly resistant to pseudomonic acid. Active fractions of pseudomonic acid-resistant IleRS (IleRS-R2) were pooled and purified to homogeneity in five further steps (Fig. 4) as described under "Experimental Procedures." The N-terminal amino acid sequence of the purified IleRS-R2 protein was determined to be STEGSGPVRFPA. By using SDS-PAGE, the molecular mass of IleRS-R2 was estimated to be 117 kDa, which was slightly higher than that of IleRS-R1 (Fig. 4, lane 6). More surprisingly, IleRS-R2 showed no inhibition in the presence of pseudomonic acid even at the concentration of 5 mM (Table I), 5 orders of magnitude greater than the Ki values observed for IleRS-R1 (Table I), and 6 orders of magnitude greater than those for E. coli IleRS (2, 25). Aminoacylation assay solution contains IleRS-R2 enzyme only at a concentration of several nanomolars, indicating that IleRS-R2 has almost no affinity for the antibiotic. So we concluded that P. fluorescens possesses two IleRSs, each with a different level of sensitivity to pseudomonic acid. This finding explains why it was difficult to determine Ki of the crude extract.
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Isolation of New ileS Gene from P. fluorescensTo clone the
novel ileS gene encoding IleRS-R2, here named ileS2, several
degenerate primers were designed from two short sequences highly conserved
among IleRSs of all the species reported to date. These sequences,
"D(C/V)WF(E/D)SG" and "Q(T/H/Y)RGWF", are adjacent to
the region of the KMSKS sequence (Fig.
5). PCR fragments of 130 bp, amplified with these primers
from P. fluorescens genomic DNA, were cloned and sequenced. In
several clones we found a DNA sequence homologous to the ileS gene,
which was 52% identical to the corresponding segment of P. fluorescens
ileS1. To our surprise, it was clear that the peptide predicted from the
DNA sequence shared some eukaryote-specific sequences conserved among
eukaryotic IleRSs (17). By
using the radiolabeled PCR fragment as a probe, Southern blot hybridization
against digested P. fluorescens DNA and cloning of the full-length
ileS2 gene were performed. The probe hybridized to an
8.5-kb
EcoRI fragment by Southern blot analysis (data not shown). On the
other hand, the DNA probe derived from ileS1 hybridized specifically
to apparently different genomic DNA fragments (data not shown). These results
indicate that two distinct genes encode the two IleRSs. An 8.5-kbp fraction of
EcoRI-digested P. fluorescens DNA was cloned into pTWV228
(Takara). One of the selected candidates obtained by colony hybridization
screening was named pR2, and 3093 bp of the structural gene was completely
sequenced to identify the entire P. fluorescens ileS2. The open
reading frame, encoding a protein consisting of 1030 amino acids, was assigned
to ileS2, because the deduced amino acid sequence of its 5'-end
following a methionine initiator perfectly matched the 12 determined
N-terminal residues of IleRS-R2 isolated from P. fluorescens. This
novel ileS gene encoded a protein with a calculated molecular weight
of 117,738, which was in good agreement with the molecular weight of 117,000
estimated by SDS-PAGE (Fig. 4,
lane 6). The G + C content of ileS2 coding sequence (59%)
and the codon usage are comparable with those of other conventional P.
fluorescens genes.
P. fluorescens IleRS-R2 Is Similar to Eukaryotic IleRSs but Not to
EubacterialsTwo consensus sequences of class I aaRSs were also
found in the P. fluorescens IleRS-R2 sequence: HYGH (HIGH homologue,
residues 5558) and KMSKR (KMSKS homologue, residues 589593).
These two regions have been proposed to form a three-dimensional structure
called a Rossman nucleotide-binding fold, made up of alternating
-strands and
-helices
(26,
27). This structure is
postulated to interact with ATP and to play a crucial role in catalysis
(2831).
Sequence comparison with the other known IleRSs revealed that the P.
fluorescens IleRS-R2 sequence was aligned well with the eukaryotic IleRS
and eukaryote-like eubacterial IleRS sequences, showing mainly 3541%
overall sequence identity as calculated by the FastA algorithm
(32). The IleRS-R2 sequence
was closely related to eukaryotic and eukaryote-like IleRSs, but not to
eubacterial and eukaryotic mitochondrial IleRS sequences, including P.
fluorescens IleRS-R1 (Fig.
5). IleRS-R2 contained representatively eukaryote-specific
(partially archaebacterial) sequences originally found in eukaryotic
cytoplasmic IleRS (Fig. 5,
green portions of amino acid residues). These sequences were well
conserved among the eukaryotic and cytoplasmic and eukaryote-like eubacterial
IleRSs described above but not among the eubacterial or eukaryotic
mitochondrial IleRSs. In this report, in order to differentiate eubacterial
IleRSs that have eukaryote-like features from eukaryotic IleRSs obtained from
eukaryotic cytoplasm, the former are designated as type C and the latter as
type E. In addition, conventional eubacterial IleRSs are designated as type B,
archaebacterial IleRSs as type A, and eukaryotic mitochondrial IleRSs as type
D. P. fluorescens IleRS-R2 is classified into type C IleRS, whereas
IleRS-R1 is designated type B IleRS based on sequence similarity
(Fig. 5).
Construction of Plasmids for Expression and Purification of IleRS ProteinsIn order to express P. fluorescens IleRS-R2 protein in E. coli cells, a full-length ileS2 gene was cloned into expression vector pEXPCR to generate plasmid pEXR2, as described previously (25) in the construction of pEXR1 containing the P. fluorescens ileS1 gene. The plasmids pEXR1 and pEXR2 were introduced into E. coli to express ileS gene products. The recombinant IleRS-R1 and IleRS-R2, as visualized by SDS-PAGE of crude cell extracts, showed molecular masses of 107 and 117 kDa, respectively. Overexpressed proteins were subsequently isolated to a single band using three chromatographic steps, as described under "Experimental Procedures" (Fig. 4, lanes 7 and 8), and were then used for preparing polyclonal antibodies. By Western blotting it was clearly confirmed that IleRS-R2 fractionated on DEAE-Sephacel column chromatography was the product of the ileS2 gene (Fig. 2). Recombinant IleRS-R2 exhibited almost the same Km values compared with those of IleRS-R2 purified from P. fluorescens (Table I). The recombinant IleRS showed little sensitivity to pseudomonic acid: 77% of the activity remained even at 1 mM pseudomonic acid, as shown in Table I (higher concentrations of the antibiotic were not tested).
Complementation StudiesP. fluorescens IleRS-R2 was able to charge isoleucine to E. coli tRNAIle in vitro (Fig. 2). To determine whether or not IleRS-R2 can also be functional in E. coli cells, the plasmid that carries the P. fluorescens ileS2 gene was introduced into E. coli ileS(ts) mutant Ts331 to see whether or not it would complement IleRS function. These mutant Ts331 cells formed colonies at a non-permissive temperature of 42 °C on an LB agar plate when transformed with plasmid containing E. coli wild-type ileS (pEXEC) (25) or plasmid containing P. fluorescens ileS1 (pEXR1) (Table III). On the other hand, the ileS2 gene-carrying plasmid (pEXR2) alone failed to restore the colony-forming ability of Ts331 cells at 42 °C even after 120 h of incubation. The IleRS-R2 protein seems to be metabolically unstable at non-permissive temperatures in in vivo situations. SDS-PAGE analysis confirmed the existence of IleRS-R2 protein in the cell extracts from E. coli Ts331 bearing pEXR2 at 30 °C (Fig. 6, lanes 5 and 6). The IleRS-R2 protein may have been denatured (and/or degraded) at 42 °C and may not have remained in the soluble fraction of crude extracts prepared after Ts331 cells expressing IleRS-R2 were grown at 30 °C for 12 h followed by 42 °C for 3 h (Fig. 6, lane 7). On the other hand, IleRS-R1 remained active, and its amount remained unchanged in the soluble fraction of crude extracts prepared from Ts331 cells expressing IleRS-R1 grown in the same conditions (Fig. 6, lane 4). These results confirmed that the ileS2 gene could be expressed in E. coli cells at permissive temperatures but that the residual activity of IleRS-R2 at non-permissive temperatures is not enough to sustain steady state growth of the Ts331 cells.
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P. fluorescens ileS2 Gene Confers Upon E. coli Cells Extreme Resistance
to Pseudomonic AcidTo test in vivo pseudomonic acid
resistance, we measured the inhibitory effect of pseudomonic acid on the
growth of E. coli DH5 cells transformed with several
IleRS-producing plasmids. The growth of cells expressing E. coli
wild-type IleRS was completely inhibited in the presence of pseudomonic acid
at 40 µg/ml (25). The
growth of cells expressing P. fluorescens IleRS-R1 was moderately
resistant to pseudomonic acid at a concentration of 40 µg/ml
(25) and was inhibited by
pseudomonic acid at 200 µg/ml (Table
II). IleRS-R2-expressing cells grew normally in the presence of as
much as 1000 µg/ml pseudomonic acid, suggesting that IleRS-R2 confers much
more resistance to E. coli cells than does IleRS-R1 or E.
coli IleRS. This apparently indicates that both P. fluorescens
IleRS-R1 and IleRS-R2 are active in vivo. E. coli strain DH5
transformed with both the ileS1 gene and the ileS2 gene
exerted pseudomonic acid resistances (>1000 µg/ml) equivalent to that of
P. fluorescens strain NCIB10586. More importantly, both P.
fluorescens IleRS-R1 and -R2, when coexpressed in E. coli cells,
can confer pseudomonic acid resistance to the host cells. Pseudomonic acid
resistance was highly elevated when both of the P. fluorescens IleRS
were expressed simultaneously in E. coli cells.
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DISCUSSION |
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IleRS-R1 and IleRS-R2 differed from each other in kinetic, chromatographic, and immunological features as well as in their sensitivity to pseudomonic acid. Why does P. fluorescens maintain two IleRS? Under laboratory conditions, P. fluorescens produces pseudomonic acid at low levels (1100 µg/ml) during the stationary growth phase and secretes the antibiotic into fermentation broth (1, 33, 34). However, their maximum production of the antibiotic in vivo is not known. The pseudomonic acid-producing bacteria may have required an IleRS that is more tolerant than the relatively sensitive IleRS-R1 to ensure that suicide would not occur in unexpected situations (e.g. in case of over-fermentation) in vivo. In addition, the bacteria may have required a more heat-stable IleRS-R1 to survive, because IleRS-R2 is metabolically unstable at relatively high temperatures. We need to disrupt each ileS gene to clarify their physiological roles in P. fluorescens. Inactivation of ileS2, leading to its killing by pseudomonic acid production, would prove the hypothesis that the IleRS-R2 is there to prevent killing.
Two P. fluorescens IleRSs share only 29.6% identical residues, suggesting that they are evolutionarily distinct from each other. In general, prokaryotes do not have more than one gene encoding for the different aaRSs specific to the same amino acid, although there are a few exceptions for two genes coexisting in the same organism. Two aaRSs encoded by distinct genes are classified into one eubacterial class in which the two show high homology, or they are classified into different eubacterial but not into archaeal/eukaryotic classes (e.g. E. coli lysS-lysU (3537), Bacillus subtilis thrS-thrZ (38), Streptomyces coelicolor trpS1-trpS2 (39), and B. subtilis tyrS-tyrZ (40)). There also are very few cases in which two enzymes are classified into evolutionarily distinct types. One of the two aaRSs belongs to the eubacterial class and the other to the archaeal class (e.g. Thermus thermophilus AspRSs (41), Synechocystis, Bacillus, and Aquifex HisRSs (42)). Besides the chromosomally encoded eubacterial IleRS, S. aureus possesses plasmidic eukaryote-like IleRS, which is resistant to pseudomonic acid (21). These cases represent examples of one organism having both eubacterial and archaeal/eukaryotic types of enzymes. The case of the P. fluorescens IleRS clearly belongs to the latter category, i.e. one prokaryote cell has evolutionarily distinct aaRSs.
Generally, it has been shown that eukaryotic (type E and type C) IleRS are more resistant to pseudomonic acid than are type A and type B IleRS (Table I) (2, 5, 17, 20, 24). Many aaRSs showing eukaryote-like features (type C) have been found recently in several bacteria (12, 42, 43). To date, including S. aureus and P. fluorescens, more than 20 bacteria that possess type C IleRSs has been reported. Although we have not tested the idea thus far, such eukaryotic features strongly suggest that all of these organisms are also resistant to pseudomonic acid. The phylogenetic classification of Mycobacterium tuberculosis IleRS as a eukaryote-like type has been reported, along with the extreme pseudomonic acid resistance of that for IleRS (17). Once the structural basis of the mechanisms for high level pseudomonic acid resistance is identified, it may provide a clue to elucidate the mechanism of resistance to pseudomonic acid possessed by many IleRSs showing eukaryotic features.
A pseudomonic acid-resistant E. coli mutant PS102 carried a single point mutation in ileS, in the IleRS of which phenylalanine was substituted for leucine at the residue of 594 (Fig. 5, highlighted by the asterisk at the bottom of the sequence alignments) (25), and the mutant showed moderate resistance to pseudomonic acid in vivo (150200 µg/ml). Crystallographic studies of S. aureus IleRS and T. thermophilus IleRS complexed with pseudomonic acid support the idea that Phe594 seems to be involved mainly in pseudomonic acid binding (23, 24). Ki values for P. fluorescens IleRS-R2 must provide resistance that is another 5 or 6 orders of magnitude higher than those for eubacterial E. coli and P. fluorescens IleRS-R1 enzymes (Table II). Considering that the single amino acid change caused only a severalfold increase in the Ki value (25), further mutations are necessary to acquire much more drastic resistance in IleRS (i.e. to increase the Ki of IleRS).
Sequence alignments show that contact residues with pseudomonic acid are highly conserved among almost all IleRS (Fig. 5) (23, 24). We can therefore rule out the possibility that these residues might explain the differences in antibiotic affinities. Pseudomonic acid has been hypothesized to be an analogue of IleRS small substrates or intermediate isoleucyl-AMP (2, 4, 23, 25). The mode of action of pseudomonic acid has been compared in detail with those of isoleucyl-AMP analogues (49). Recent crystallographic analysis of T. thermophilus IleRS complexed with the isoleucyl-AMP analogue suggested that the antibiotic blocks the binding of active intermediate isoleucyl-AMP (24). Resistance to pseudomonic acid in IleRS (i.e. to increase the Ki of IleRS) may generally be acquired by substitutions that reduce the affinity to pseudomonic acid but maintain affinity to IleRS substrates and to isoleucyl-AMP.
This study has shown that there are several "eubacterial species" that contain "eukaryotic" (more accurately, eukaryote-like) aaRSs. So, these aaRS sequences cannot be classified simply into three taxonomic types (eubacterial, archaebacterial, and eukaryotic). The mosaic features of the eubacterial/eukaryotic type of bacterial aaRSs may be caused by the transfer of genes beyond eubacteria and eukaryote lineages. Moreover, it should be noted that among cases of eubacterial cells possessing eukaryote-like aaRSs, the eukaryote-like synthetase seems to be a single enzyme, with no eubacterial counterpart that is specific for the same cognate amino acid found in the eubacterial cell. M. tuberculosis has a single type C IleRS but lacks the conventional eubacterial type B enzyme. Similarly, a single type C IleRS was found in many bacteria. Shiba et al. (12) and Doolittle and Handy (16) proposed that a single horizontal gene transfer from eukaryote to eubacterium, followed by divergence among eubacteria and a possible displacement of the conventional eubacterial enzyme by the eukaryotic version, may have led to two kinds of bacterial IleRSs families with different origins. Importantly, such a gene replacement seems not to have occurred in S. aureus and P. fluorescens.
It appears that particular strains of P. fluorescens possess the eukaryotic ileS gene.2 This finding supports the gene mobilization hypothesis. If the horizontal transfer of ileS2 is true, when did that event occur? The presence of the S. aureus plasmid carrying a eukaryotic ileS gene provides a candidate for recent horizontal transfer. Unlike the case of S. aureus, the eukaryotic ileS2 gene of P. fluorescens is supposed to be present on its chromosome, according to the results of Southern blotting, plasmid extraction, and cured cell experiments (data not shown). The eukaryotic ileS gene might have been transferred to an ancestor strain of P. fluorescens at some point prior to the recent gene transfer event in S. aureus. The eukaryotic gene has been rearranged and merged into the P. fluorescens chromosome over a long period of evolution. The possibility of horizontal transfers of a pseudomonic acid resistance gene from early eukaryotes to prokaryotes has been proposed (44). That study posited that a eukaryotic ileS gene was first transferred to an unknown bacterium after the divergence of Eukarya and Archaea and was then transferred to several species of bacteria. Although we cannot confirm whether the ancestral P. fluorescens was the first bacterium to acquire the eukaryotic ileS gene, we can easily guess that this producer of pseudomonic acid would have required a pseudomonic acid-insensitive IleRS by the time that antibiotic pseudomonic acid was first produced.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Ministry of
Education, Science, Sports, and Culture of Japan. The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Human Health and Sciences, Sendai Shirayuri
Women's College, Izumi, Sendai 981-3107, Japan.
To whom correspondence should be addressed: RIKEN Genomic Sciences Center,
Suehiro-cho 1-7-22, Tsurumi, Yokohama 230-0045, Japan. Tel.: 81-3-5841-4394;
Fax: 81-3-5841-8057; E-mail:
yanagi{at}gsc.riken.go.jp.
1 The abbreviations used are: IleRSs, isoleucyl-tRNA synthetases; aaRS,
aminoacyl-tRNA synthetase; DTT, dithiothreitol.
2 T. Yanagisawa, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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