How does Pseudomonas fluorescens avoid suicide from its antibiotic pseudomonic acid?: Evidence for two evolutionarily distinct isoleucyl-tRNA synthetases conferring self-defense.

Two isoleucyl-tRNA synthetases (IleRSs) encoded by two distinct genes (ileS1 and ileS2) were identified in pseudomonic acid (mupirocin)-producing Pseudomonas fluorescens. The most striking difference between the two IleRSs (IleRS-R1 and IleRS-R2) is the difference in their abilities to resist pseudomonic acid. Purified IleRS-R2 showed no sensitivity to pseudomonic acid even at a concentration of 5 mm, 105 times higher than the Ki value of IleRS-R1. The amino acid sequence of IleRS-R2 exhibits eukaryotic features that are originally found in eukaryotic proteins. Escherichia coli cells transformed with the ileS2 gene exerted pseudomonic acid resistance more than did those transformed with ileS1. Cells transformed with both genes became almost as resistant as P. fluorescens. These results suggest that the presence of IleRS-R2 could be the major reason why P. fluorescens is intrinsically resistant to the antibiotic. Here we suggest that the evolutionary scenario of the eukaryotic ileS2 gene can be explained by gene acquisition and that the pseudomonic acid producer may have maintained the ileS2 gene to protect itself from pseudomonic acid.

Pseudomonic acid (mupirocin) is an antibacterial agent produced by Pseudomonas fluorescens NCIB10586 (1). This antibiotic is known as a potent inhibitor of many bacterial isoleucyl-tRNA synthetases (IleRSs). 1 It competitively inhibits IleRS with respect to isoleucine (2), thereby arresting protein synthesis. The inhibitory constants (K i ) for prokaryotic (eubacterial and archaebacterial) IleRSs in in vitro aminoacylation reaction are generally 10 Ϫ8 to 10 Ϫ9 M (2)(3)(4). Meanwhile, this antibiotic has little or no potency toward eukaryotic IleRSs (2,5). This striking specificity in the mode of action and in its unique structure place pseudomonic acid into an unusual class of antibacterial agents (6,7) and have led to the clinical use of pseudomonic acid as a topical antibacterial agent to prevent Staphylococcus aureus infection (8,9).
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 selfdefense 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.

EXPERIMENTAL PROCEDURES
Materials, Enzymes, and Chemicals-Biochemical and molecular biological procedures were performed using commercially available enzymes, chemicals, and other materials. The pseudomonic acid was a gift from SmithKline Beecham Pharmaceuticals (Betchworth, Surrey, UK).
Culture Media and Growth Conditions-E. 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 Studies-Aminoacylation activi-* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank TM /EBI Data Bank with accession number(s) AB062785.
Preparation of Crude Cell Extract-P. fluorescens cells were cultured in LB medium (10 ml) at 26°C for 15 h. The cells were harvested by centrifugation at 12,000 ϫ 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 ϫ g for 15 min. The supernatant was further ultracentrifuged at 165,000 ϫ 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 Chromatography-P. fluorescens NCIB10586 cells were cultured in LB medium (1 liter) at 26°C for 12 h. Cells were harvested by centrifugation at 10,000 ϫ g, and the pellet was suspended in buffer A5. The suspension was sonicated, and the cell debris was pelleted by centrifugation at 10,000 ϫ g for 10 min. Subsequently, the supernatant was ultracentrifuged at 100,000 ϫ 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 ϫ g for 20 min. The precipitate was resuspended in buffer A5, ultrasonicated, and ultracentrifuged at 100,000 ϫ 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) 2 SO 4 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) 2 SO 4 . After the column was washed with buffer A1 containing 1 M (NH) 2 SO 4 , proteins were eluted with a linear gradient of 1 to 0 M (NH) 2 SO 4 . 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 Ϫ20°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" (5Ј-GAYGTNTGGTTYGANTSNGG-3Ј named V1 (where Y is C ϩ T, S is G ϩ C, and N is A ϩ G ϩ C ϩ T), and "DCWFESG" (5Ј-GAYTGYTGGTTYGAYTSNGG-3Ј named C1). Downstream set of the two primers were from "QTRGWFY" (5Ј-ARAACCANCCNCKNGT-RTG-3Ј named T1; K is G ϩ T) and from "DQ(H/Y)RGWF" (5Ј-AAC-CANCCNCKRTRYTGRTC-3Ј named YH1; R is A ϩ 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 ϳ130 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.
DNA Probes-133-bp DNA fragment, amplified using C1 and T1 primers, was gel-purified and radiolabeled with [␣-32 P]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 Gene-For cloning, EcoRI-digested P. fluorescens DNA was fractionated by agarose gel electrophoresis. An ϳ8.5-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 ϳ1000 bp of the flanking region, was completely sequenced.
Western Blot Analysis-Each 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. coli-The construction of plasmid pEXR1 (the former name is pPFNB7) was described previously (25). To construct pEXR2, the P. fluorescens ileS2 gene with PCR-modified 5Ј-and 3Ј-ends was ligated into the NdeI/ HindIII double-digested pEXPCR expression vector. The primers used were 5Ј-GACAGGTGTGACAT(ATG)AGTACGGAAGGAAGTGGGCC-3Ј and 5Ј-ACCAGTCGGCAAGCTT(TCA)GGCCAGTACGCTACGGCGC-3Ј (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 g/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 g/ml antipine, 2 g/ml leupeptin, 1 g/ml pepstatin, and 1 g/ml chymostatin. The cells were broken by sonication, and crude extracts were centrifuged at 30,000 ϫ g for 20 min. The supernatants were subjected to ultracentrifugation at 100,000 ϫ 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 Il-eRS-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 Analysis-E. 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 Resistance-Pseudomonic acid was diluted by 0.8% LB agar media (100 l) at 10, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 600, 800, and 1000 g/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 ϫ 10 9 cells/ml by the LB medium, and then 2-l spots (10 6 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.

RESULTS
Pseudomonic Acid Sensitivity of Crude Cell Extract from P. fluorescens-How does P. fluorescens, producer of the antibiotic pseudomonic acid, avoid suicide? To clarify the precise mechanism of its self-defense, we kinetically analyzed the IleRS activity in crude cell extract from P. fluorescens strain NCIB10586. The K m for isoleucine was determined to be 10.4 M, but the K m for ATP was observed as biphasic in the Lineweaver-Burk plot and measured as two Michaelis constants (15.2 and 142.9 M). We could not calculate the K i value for pseudomonic acid precisely from its inhibition curves (Fig. 1). At 4 mM ATP, 22.7% of the total aminoacylation activity was drastically inhibited in the presence of as little as 5 M pseudomonic acid. But the remaining activity could not be abolished; even when the antibiotic was increased to 1 mM, 64.9% of the total activity remained (Fig. 1A). When the activity was measured at 1 mM ATP, 32.1% of the enzyme activity was drastically inhibited in the presence of 100 nM pseudomonic acid, but 45.5% of the activity remained even in the presence of 1 mM pseudomonic acid (Fig. 1B). So we kinetically analyzed the purified IleRS-R1 (the previously reported IleRS) (25) to find that K i values for pseudomonic acid with respect to isoleucine and ATP were calculated as an order of 10 Ϫ8 M (Table I). It was curious that the K i values were considerably lower than those measured with crude extract reported so far (14.5 mM) (18). Moreover, the K i values of IleRS-R1 appeared not to be able to explain why P. fluorescens is resistant to high concentrations of pseudomonic acid (Ͼ1000 g/ml) (Tables I and II). From these results, we hypothesized that the crude extract from the pseudomonic acid-producing bacteria contains more than one IleRS.
Evidence for Two IleRSs Conferring Distinct Pseudomonic Acid Resistance-This 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 M 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 K i 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 K i of the crude extract.
Isolation of New ileS Gene from P. fluorescens-To 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 ϳ130 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 ϳ8.5-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 5Ј-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 ϩ 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 Eubacterials-Two consensus sequences of class I aaRSs were also found in the P. fluorescens IleRS-R2 sequence: HYGH (HIGH homologue, residues 55-58) and KMSKR (KMSKS homologue, residues 589 -593). 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 (28 -31). 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 35-41% 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 Proteins-In 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 prod-    ucts. 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 K m 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 Studies-P. fluorescens IleRS-R2 was able to charge isoleucine to E. coli tRNA Ile 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 FIG. 5. Sequence alignments of P. fluorescens IleRS-R2 with other IleRS. The sequences were aligned by using the ClustalW program (47), and parts of these alignments were optimized and adjusted manually. Eubacterial IleRS that have eukaryote-like features are categorized as type C (light green blocks designated as C beside the sequence alignments), whereas eukaryotic IleRS obtained from eukaryotic cytoplasm are categorized as type E (green blocks designated as E). Conventional eubacterial IleRS are categorized as type B (yellow blocks designated as B), archaebacterial IleRS as type A (red blocks designated as A), and eukaryotic mitochondrial IleRS as type D (orange blocks designated as D). Highly conserved amino acid residues among many IleRS are shown in red, and similar amino acids are shown in orange. Conserved residues among eukaryotic (partially archaebacterial and eubacterial) IleRS are shown in green. Conserved residues among eubacterial and archaebacterial (partially eukaryotic) IleRS are shown in light blue. The KMSKS sequence is highlighted on the top line. At the residue of 594 in PS102 IleRS, phenylalanine substituted for leucine is highlighted as an asterisk on the bottom line. Dashes represent breaks in actual amino acid sequences of respective proteins to allow sequence alignment with IleRS-R2. Thick red arrows above the alignment indicate the amino acid residues from which degenerate primers were designed and the direction of the primers (see "Experimental Procedures"). Numbers at the top correspond to the amino acid residues of P. fluorescens IleRS-R2, and those at the bottom correspond to those of E. coli IleRS. Each accession number is shown beside the sequence alignment. 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.
P. fluorescens ileS2 Gene Confers Upon E. coli Cells Extreme Resistance to Pseudomonic Acid-To 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 g/ml (25). The growth of cells expressing P. fluorescens IleRS-R1 was moderately resistant to pseudomonic acid at a concentration of 40 g/ml (25) and was inhibited by pseudomonic acid at 200 g/ml (Table  II). IleRS-R2-expressing cells grew normally in the presence of as much as 1000 g/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 (Ͼ1000 g/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. DISCUSSION We isolated and characterized a novel IleRS (IleRS-R2) from pseudomonic acid producing P. fluorescens. The K i value of purified IleRS-R2 appears to be at least 10 5 -to 10 6 -fold greater than those of either type A IleRS or type B IleRS (Table I) (2)(3)(4). We demonstrated that P. fluorescens has two distinct IleRS (IleRS-R1 and IleRS-R2) that are encoded by two distinct ileSs. E. coli cells containing both ileS genes exerted as much pseudomonic acid resistance as P. fluorescens did. These results explain why P. fluorescens can grow freely in medium containing more than 1000 g/ml of the antibiotic (Table II). Tables I and II raise the issue that, despite a million-fold difference in K i values between ileS1 and ileS2, the largest measurable difference in pseudomonic acid sensitivities in vivo is about 5-fold. It is possible that factors other than enzyme sensitivity determine cellular sensitivity. For example, permeation of the antibiotic into the cell membrane may be the cause of the disparity between K i values of IleRS enzymes and their cellular resistances in vivo.
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 (1-100 g/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 resi- c Plasmid pEXR2 failed to restore the colony-forming ability of Ts331 cells, which may be due to the metabolical instability of IleRS-R2 protein at 42°C (Fig. 6).
FIG. 6. P. fluorescens IleRS-R2 is metabolically unstable at non-permissive temperatures. S100 supernatants were prepared from Ts331(pEXR1) cells and from Ts331(pEXR2) cells after the treatment at 42°C and were subjected to SDS-PAGE analysis. S100 supernatant was then extracted from the following cells: dues, 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 (35)(36)(37), 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 (150 -200 g/ml). Crystallographic studies of S. aureus IleRS and T. thermophilus IleRS complexed with pseudomonic acid support the idea that Phe 594 seems to be involved mainly in pseudomonic acid binding (23,24). K i 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 K i value (25), further mutations are necessary to acquire much more drastic resistance in IleRS (i.e. to increase the K i 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 K i 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, eukaryotelike) 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.