Abstract
LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase] is a metalloamidase that catalyzes the first committed step in the biosynthesis of the lipid A component of lipopolysaccharide. A previous study (H. R. Onishi, B. A. Pelak, L. S. Gerckens, L. L. Silver, F. M. Kahan, M. H. Chen, A. A. Patchett, S. M. Galloway, S. A. Hyland, M. S. Anderson, and C. R. H. Raetz, Science 274:980-982, 1996) identified a series of synthetic LpxC-inhibitory molecules that were bactericidal for Escherichia coli. These molecules did not inhibit the growth of Pseudomonas aeruginosa and were therefore not developed further as antibacterial drugs. The inactivity of the LpxC inhibitors for P. aeruginosa raised the possibility that LpxC activity might not be essential for all gram-negative bacteria. By placing the lpxC gene of P. aeruginosa under tight control of an arabinose-inducible promoter, we demonstrated the essentiality of LpxC activity for P. aeruginosa. It was found that compound L-161,240, the most potent inhibitor from the previous study, was active against a P. aeruginosa construct in which the endogenous lpxC gene was inactivated and in which LpxC activity was supplied by the lpxC gene from E. coli. Conversely, an E. coli construct in which growth was dependent on the P. aeruginosa lpxC gene was resistant to the compound. The differential activities of L-161,240 against the two bacterial species are thus the result primarily of greater potency toward the E. coli enzyme rather than of differences in the intrinsic resistance of the bacteria toward antibacterial compounds due to permeability or efflux. These data validate P. aeruginosa LpxC as a target for novel antibiotic drugs and should help direct the design of inhibitors against clinically important gram-negative bacteria.
Lipopolysaccharide has a critical function in gram-negative bacterial membrane integrity and resistance to host defenses, and therefore, the conserved lipopolysaccharide biosynthetic enzymes are attractive targets for novel antibacterial drugs. A drug targeting enzymes of this biosynthetic pathway would need to be active against Pseudomonas aeruginosa and other nonfermenting gram-negative bacterial species, as well as against Escherichia coli and other enteric bacteria, to be clinically useful. The P. aeruginosa outer membrane is less permeable to small molecules than that of E. coli, and P. aeruginosa has several multidrug efflux pumps. As a result of both of these factors, P. aeruginosa is less susceptible than E. coli to many antibiotics (24). Several laboratories have focused on the metalloenzyme LpxC [UDP-(3-O-acyl)-N-acetylglucosamine deacetylase], since it catalyzes the first committed step in lipid A synthesis (Fig. 1) and has been demonstrated to be essential for the growth of E. coli (3, 12, 38). P. aeruginosa LpxC is similar in sequence (Fig. 2) and catalyzes the same activity (11). While the essentiality of LpxC activity for P. aeruginosa has not been formally proven, the lpxC gene was not inactivated in a saturating transposon mutagenesis study (15). These data suggest that it might be possible to discover LpxC inhibitors active against both E. coli and P. aeruginosa. However, none of the early LpxC inhibitors, some of which showed antibacterial activity against E. coli and certain other organisms, were able to inhibit growth of P. aeruginosa (5, 12, 27-29). It was tempting to assume that the reason for this failure was the intrinsic resistance of P. aeruginosa to antibiotics. Challenging this assumption, we undertook the studies described here to evaluate the basis for the refractory nature of P. aeruginosa to LpxC inhibitors that are effective against E. coli. We focused on the compound L-161,240 (Fig. 1), the most active of the LpxC inhibitors reported by researchers at Merck (4, 27). We found that the critical reason for the inactivity of this compound against P. aeruginosa was its failure to inhibit enzyme activity. These findings have implications for designing effective strategies to identify LpxC inhibitors that can be developed as novel antibacterial drugs.
FIG. 1.
(A) LpxC catalyzes the deacetylation of UDP-(3-O-acyl)-N-acetylglucosamine, the second step in the biosynthesis of lipid A. Because the first step is reversible, the LpxC reaction is the first committed step in the conversion of UDP-N-acetylglucosamine to lipid A. (B) Structure of the LpxC inhibitor L-161,240 (27).
FIG. 2.
Comparison of predicted LpxC sequences from E. coli and P. aeruginosa. An 82% similarity and 57% identity is shared between the two sequences over the entire length of the protein. Key residues are indicated by color and also by symbols above the residues, as follows. The novel zinc binding motif characteristic of all known LpxC enzymes, HKXXD, is shown in red (stars). Four other conserved histidines are shown in blue (▪), and two E. coli histidines that are absent from P. aeruginosa are shown in cyan (□). The conserved phenylalanines providing a hydrophobic patch (7) are shown in orange (▴). Analogous to A. aeolicus, in E. coli histidine 265 may stabilize the oxyanion intermediate of the transition state. Significant differences in enzyme fold or participating residues are suggested by the differences in key residues, for example, the two nonconserved histidines in E. coli.
MATERIALS AND METHODS
Reagents and bacterial cultivation.
The bacterial strains and plasmids used are listed in Table 1. P. aeruginosa strains were grown at 37°C in Luria-Bertani (LB) broth (Difco) or plated on sheep blood agar (Remel). E. coli was grown in LB broth or on LB agar. EDTA, bis-Tris buffer, sucrose, arabinose, and dimethyl sulfoxide (DMSO) were purchased from Sigma as ultrapure agents. Yeast extract and tryptone were obtained from Difco. Restriction enzymes, T4 DNA ligase, and their reaction buffers were obtained from New England Biolabs. Polymyxin B nonapeptide, tetracycline, ampicillin, carbenicillin, gentamicin, and kanamycin were all purchased from Sigma. Compound L-161,240 was synthesized as described previously (4). Antibacterial compounds were dissolved in DMSO to make stock solutions of polymyxin B nonapeptide at 3 mg/ml, L-161,240 at 10 mg/ml, and tetracycline at 125 mg/ml. For growth curves, DMSO was added to control tubes as needed so that DMSO concentrations were the same in all cultures within each experiment.
TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Relevant characteristic(s)a | Source or reference(s) |
|---|---|---|
| P. aeruginosa strains | ||
| PAO1 | Wild type; sequenced strain | 10, 35 |
| PAO200 | mexAB-oprM | 32 |
| ATCC 35151 (Z61) | Chemically mutagenized strain hypersusceptible to antibiotics | 18, 39 |
| PAO1-PBAD-lpxC | lpxC promoter replaced by araBAD promoter | This work |
| E. coli strains | ||
| W3110 | K-12 derivative | E. coli Genetic Stock Center |
| DH5α | Cloning host | Gibco/Life Technologies |
| BL221/DE3/pLysS/pJEJ1 | T7 RNA polymerase-driven expression system for E. coli lpxC | 13 |
| JBK-1/pKD6 | Chromosomal lpxC disrupted with Km cassette; lpxC on temp-sensitive plasmid; Amp | 34 |
| Plasmids | ||
| pET21b | Expression plasmid; Amp | Novagen |
| pDN19 | Low-copy-number plasmid; Tet | 25 |
| pUCP26 | Tet | 26 |
| pUCP30T | Replicates in both E. coli and P. aeruginosa; Gm | 33 |
| pSP72 | Amp | Promega |
| pBAD/His B | araC+; ParaBAD | Invitrogen |
| pEC-lpxC1 | lpxC from E. coli cloned into pDN19 | This work |
| pPA-lpxC1 | lpxC from P. aeruginosa cloned into pDN19 | This work |
| pEC-lpxC2 | lpxC from E. coli cloned into pUCP30T | This work |
| pPA-lpxC2 | lpxC from P. aeruginosa cloned into pUCP30T | This work |
| pPW101 | Derived from pSP72; oriT+ | This work |
| pBEM10 | Derived from pPW101; recombination with P. aeruginosa chromosome results in placing lpxC under control of ParaBAD; Amp; Tet | This work |
Antibiotic resistance markers: Amp, ampicillin; Gm, gentamicin; Km, kanamycin; Tet, tetracycline.
Enzyme inhibition assays.
LpxC activity was measured as previously described (13, 20), using either crude cell extracts (38) of E. coli W3110 or P. aeruginosa PAO1 or purified enzyme from E. coli BL21/DE3/pLysS/pJEJ1 (14) or P. aeruginosa PAO1 (16) as the enzyme source. Assays were done in 25 mM phosphate buffer at pH 7.4 with 5 μM substrate at 30°C, with enzyme concentrations (typically 0.5 to 10 nM) adjusted to keep the conversion below 10% over the time course of the assays.
DNA manipulations.
Standard recombinant DNA procedures were used (30). The primers for amplification of the coding region of the lpxC genes included NdeI and EcoRI restriction sites for subsequent cloning. For the E. coli gene, the primers were 5′-GGGAATTCCATATGATCAAACAAAGGACACTTAAACGT-3′ and 5′-CCGGAATTCTTATGCCAGTACAGCTGAAGGCGCT-3′, and for the P. aeruginosa gene, they were 5′-GGGAATTCCATATGATGATCAAACAACGCACCTTGAAGAACAT-3′ and 5′-CCGGAATTCCTACACTGCCGCCGCCGGGCGCATATAG-3′. These primers were used in a PCR mixture containing as the template either 10 to 50 μg P. aeruginosa genomic DNA or 1 μg plasmid pKD6 containing the E. coli lpxC gene (34). The lpxC genes were amplified using Pwo DNA polymerase (Roche) in a 100-μl reaction mixture containing a 200 μM concentration of each deoxynucleoside triphosphate and a 0.5 μM concentration of each primer for 30 cycles (94°C denaturation, 55°C annealing, and 72°C polymerization). The PCR products were purified with the QIAquick PCR purification kit from QIAGEN and digested with NdeI and EcoRI restriction enzymes. The bands of the sizes predicted for the lpxC genes were identified following gel electrophoresis and excised from the gel. The excised DNA was purified using the QIAquick gel extraction kit from QIAGEN. The purified DNA was cut with NdeI and EcoRI and ligated into the T7 expression vector (36) pET21b (Novagen) that had been cut in the multiple cloning site with NdeI and EcoRI. The ligation mixture was transformed into DH5α, which was plated on LB agar containing ampicillin (250 μg/ml). The inserts in the resulting clones were sequenced before being subcloned. The E. coli and P. aeruginosa inserts were subcloned into pDN19 to produce plasmids pEC-lpxC1 and pPA-lpxC1, respectively, for low-copy-number complementation of E. coli JBK-1 and subcloned into pUCP30T to produce plasmids pEC-lpxC2 and pPA-lpxC2, respectively, for complementation of the P. aeruginosa promoter replacement mutation.
Construction of a P. aeruginosa strain with lpxC expression tightly controlled by the araBAD promoter.
Promoter replacement was carried out using a homologous recombination strategy, whereby recombination of pBEM10 with P. aeruginosa removed the native lpxC promoter and placed the tightly regulated araBAD promoter upstream of lpxC on the chromosome (Fig. 3). In preliminary experiments (data not shown) in which this promoter controlled expression of the luciferase gene lux (9), it was found that in P. aeruginosa, there was a low background level of expression in the absence of arabinose and that this expression was not eliminated by the addition of glucose as it is in E. coli (22). Background levels of promoter expression were successfully reduced by altering the sequence of the ribosome binding site from its original sequence of AGGAG to CTTCT.
FIG. 3.
Construction of PAO1-PBAD-lpxC, a P. aeruginosa mutant in which lpxC is controlled by the araBAD promoter. (A) The promoter replacement vector pBEM10 was derived from pPW101 and PCR products of the 5′ region of P. aeruginosa lpxC as described in Materials and Methods. (B) Promoter replacement mutagenesis. Transformation of pBEM10 into P. aeruginosa removed the native lpxC promoter and replaced it with the tightly regulated araBAD promoter just upstream of the chromosomal copy of lpxC. lpxC expression occurs only in the presence of arabinose.
Plasmid pPW101 was constructed by ligating the RP4 origin of transfer oriT into pSP72. oriT was amplified from plasmid pEX100T (33) with an introduction of NdeI and AatII restriction sites. To create pBEM10, the following DNA pieces were amplified and sequentially ligated into pPW101: the tetracycline resistance marker from plasmid pUCP26 (26), the araBAD promoter (8) from the plasmid pBAD HisB (Invitrogen), with an altered ribosome binding site, the araC repressor gene (17, 31), also from pBAD HisB, and the first 340 base pairs of P. aeruginosa lpxC gene PA4406. The tetracycline resistance marker was amplified using a forward primer that introduced a BglII site (5′-AGATCTCAAGGGTTGGTTTGCGCA-3′) and a reverse primer that introduced an EcoRI site (5′-GAATTCTAATTCTCATGTTTGACA-3′). The araBAD promoter and araC gene were amplified as one piece from the pBAD HisB vector. The forward primer introduced an XhoI site (5′-CTCGAGGCATGCATAATGTG CCTGTC-3′), and the reverse primer introduced a HindIII site (5′-AAGCTTCTCCTGTTAGCCCAAAAAAACG-3′). A primer set was used to alter the ribosome binding site and introduce an upstream BssHII site (5′-GCGCGCGGACGAAAGTAAACCCACTGG-3′) and a downstream HindIII site (5′-AAGCTTATTCAGAAGGTTAGCCCAAAAAAACGGG-3′). The first 340 bases of PAO1 lpxC were amplified from PAO1 genomic DNA. The forward primer introduced a HindIII site (5′-AAGCTTATGATCAAACAACGCACCTT-3′), and the reverse primer introduced an XbaI site (5′-TCTAGAAGCGCTGCCATCCATGATCGG-3′). These pieces were then ligated into pPW101 to form the final product, pBEM10. Transformation of pBEM10 into P. aeruginosa removed the native lpxC promoter and replaced it with the tightly regulated araBAD promoter just upstream of the chromosomal copy of lpxC. Growth of the resulting strain, designated PAO1-PBAD-lpxC, was dependent on arabinose.
Growth curves.
Bacterial cultures were prepared by diluting overnight cultures to an optical density at 600 nm (OD600) of 0.1 in 5 ml of LB. The inhibitor L-161,240 was added to the bacterial cultures to a final concentration of 50 μg/ml or 10 μg/ml. The cultures were incubated with shaking, and 0.8 ml was taken for OD600 readings over the course of the experiment. DH5-α, PAO1, and PAO200 were all grown at 37°C. In the cases where temperature-sensitive JBK-1 strains were being assayed, the cultures were grown at 42°C for both the overnight and the time course cultures.
Microtiter assay for growth inhibition of promoter replacement mutants by L-161,240.
Single colonies of DH5α, PAO1, and each mutant strain were picked and grown in LB at 37°C, with shaking for approximately 4 h, and then each culture was diluted to 5 × 105 cells/ml. A total of 200 μl of each diluted culture was added to wells containing 4 μl of inhibitor. The 96-well plates were incubated at 37°C overnight and their OD600s were determined using the Spectramax Plus plate reader (Molecular Devices).
RESULTS
Compound L-161,240, the most potent of the LpxC inhibitors described by Onishi et al., is active against E. coli, with an MIC of 1 μg/ml, but has no activity against P. aeruginosa (MIC > 50 μg/ml) (27). We found that this compound was inactive against wild-type strains of P. aeruginosa (PAO1 and ATCC 27853), strain PAO200, in which genes mexAB and oprM, encoding the major multidrug efflux pump, are deleted (32), and the hypersusceptible strain Z61 (ATCC 35151) (2). Treatment of PAO1 and PAO200 with polymyxin B nonapeptide (3 μg/ml) to increase the permeability of the membrane (23) failed to render these strains susceptible to L-161,240 (data not shown). These observations suggested that the failure of L-161,240 to reach its target within P. aeruginosa cells might not be the primary reason for its failure to inhibit growth of P. aeruginosa.
L-161,240 was 38 times more potent toward E. coli than toward P. aeruginosa in an in vitro assay of the LpxC activity in crude bacterial extracts (Table 2). For E. coli, the inhibitor was equally as active toward purified LpxC as it was toward the LpxC in a bacterial extract. However, for P. aeruginosa, purification of LpxC increased its susceptibility to L-161,240.
TABLE 2.
L-161,240 inhibition of the LpxC enzyme from either purified or crude sourcesa
| Enzyme source | IC50 (μM) of L-161,240 against:
|
IC50 ratio (P. aeruginosa/ E. coli) | |
|---|---|---|---|
| E. coli DH5α | P. aeruginosa PAO1 | ||
| Crude extracts | 0.037 ± 0.002 | 1.40 ± 0.07 | 37.8 |
| Purified enzyme | 0.023 ± 0.003 | 0.22 ± 0.03 | 9.6 |
IC50, 50% inhibitory concentration. Data are means ± standard deviations from three replicate assays.
These observations indicate that P. aeruginosa LpxC is more resistant to L-161,240 than the LpxC from E. coli and that reducing the effect of intrinsic resistance mechanisms does not render P. aeruginosa susceptible to growth inhibition by L-161,240. These data do not allow us to determine the relative contributions of these two aspects of P. aeruginosa's resistance to the compound. In order to assess the effect of L-161,240 on P. aeruginosa LpxC in a bacterial cell that is known to allow entry of the compound, we made use of an E. coli construct in which growth at 42°C was dependent on the presence of a functional lpxC gene from either E. coli or P. aeruginosa. The chromosomal lpxC gene of E. coli strain JBK-1/pKD6 has been inactivated; a wild-type copy of E. coli lpxC is provided on the temperature-sensitive replicon pKD6, which also confers ampicillin resistance (34). Since lpxC is essential for growth, this strain is not viable at 42°C because the functional copy is on the temperature-sensitive replicon. We transformed JBK-1/pKD6 with a low-copy, non-temperature-sensitive replicon carrying wild-type lpxC from either E. coli or P. aeruginosa. Selection for tetracycline resistance at 42°C yielded ampicillin-susceptible transformants with either the E. coli or the P. aeruginosa construct, designated JBK-1/pEClpxC1 and JBK-1/pPAlpxC1, respectively. This result indicated that lpxC from P. aeruginosa could be expressed in the E. coli background and was capable of substituting for the inactivated chromosomal copy.
When incubated at 42°, JBK-1/pEC-lpxC1 failed to grow in the presence of the LpxC inhibitor L-161,240, as expected. However, the isogenic strain JBK-1/pPA-lpxC1 was resistant to the LpxC inhibitor at this temperature (Fig. 4). This indicated that the relative insensitivity of the P. aeruginosa enzyme to the inhibitor is sufficient to confer resistance to growth inhibition.
FIG. 4.
The lpxC gene from P. aeruginosa confers L-161,240 resistance to E. coli lacking a functional endogenous lpxC gene. The strain JBK-1/pKD6 contains the chromosomal lpxC gene disrupted with a kan element and a wild-type copy of lpxC on the temperature-sensitive replicon pKD6. In strains JBK-1/pEC-lpxC1 and JBK-1/pPA-lpxC1, pKD6 has been replaced by plasmids (not temperature sensitive) bearing wild-type lpxC from E. coli and P. aeruginosa, respectively. The bacterial strains were incubated in LB medium containing 10 μg/ml L-161,240 or no drug, and bacterial growth was determined by monitoring the change in absorbance at 600 nm.
We constructed a mutant of P. aeruginosa in which the native promoter of lpxC was replaced by an inducible promoter (Fig. 3). Modification of the E. coli araBAD promoter to render it tightly regulated in P. aeruginosa is described in Materials and Methods. The resulting P. aeruginosa mutant, designated PAO1-PBAD-lpxC, was fully capable of growth in the presence of arabinose but did not grow at all in the absence of this inducer. This confirmed that P. aeruginosa is similar to E. coli in that it contains only one functional copy of lpxC and in that its activity is essential for growth. PAO1-PBAD-lpxC was transformed with a plasmid containing either P. aeruginosa lpxC or E. coli lpxC (pPA-lpxC2 or pEC-lpxC2, respectively). The transformants were then incubated in various concentrations of the LpxC inhibitor. In the absence of arabinose, P. aeruginosa strains expressing only E. coli LpxC were nearly as susceptible to the inhibitor as E. coli DH5α strains, while transformants with the lpxC gene from P. aeruginosa were resistant, as was the parent P. aeruginosa strain PAO1 (Fig. 5). When the experiment was carried out in the presence of 0.2% arabinose to induce expression of the chromosomal lpxC gene, neither transformant was susceptible to L-161,240 (Fig. 5). This confirmed that the P. aeruginosa lpxC enzyme itself is the primary factor in the resistance of P. aeruginosa to L-161,240. The intrinsic resistance mechanisms of P. aeruginosa, in the form of membrane impermeability or inhibitor efflux, play a relatively minor role.
FIG. 5.
Treatment of promoter replacement mutant of P. aeruginosa with various concentrations of the LpxC inhibitor L-161,240 in the absence (A) or presence (B) of arabinose. Regardless of the presence of arabinose, wild-type E. coli DH5α is susceptible to the inhibitor and wild-type (wt) PAO1 is resistant. PAO1-PBAD-lpxC was transformed with a plasmid carrying the lpxC gene from either E. coli (pEC-lpxC2) or P. aeruginosa (pPA-lpxC2). In the absence of arabinose (A), the chromosomal lpxC gene is not expressed and the strain carrying pEC-lpxC2 is susceptible to the inhibitor while the strain carrying pPA-lpxC2 is resistant. In the presence of 0.2% arabinose (B), the chromosomal lpxC gene in PAO1-PBAD-lpxC is expressed, conferring resistance to the LpxC inhibitor that is not affected by the plasmid-borne lpxC.
DISCUSSION
Our data indicate that the LpxC of P. aeruginosa is refractory to inhibition by compound L-161,240 at concentrations 10-fold higher than those that completely inhibit the LpxC of E. coli. This was demonstrated in both in vitro assays of enzyme activity and bacterial growth experiments using a P. aeruginosa construct in which the only active lpxC gene was from E. coli and a complementary construct in which growth of E. coli depended on the lpxC gene from P. aeruginosa. Thus, it is clearly the difference in enzymes, not differences between species in membrane structure or specificity of efflux pumps, that is the primary mediator of the differential susceptibilities of the two bacterial species to this inhibitor.
There remains, however, some cytoplasmic process in P. aeruginosa that further reduces the activity of L-161,240 (Table 2). Our experiments reveal the existence of, but do not characterize, this process. The inhibitor could be sequestered or inactivated within P. aeruginosa. Alternatively, P. aeruginosa LpxC could itself be complexed with other molecules in such a way as to limit access of the inhibitor to the enzyme. Nonetheless, the different potencies of L-161,240 against the two enzymes appear to be the single factor that contributes most to the lack of activity of the inhibitor against P. aeruginosa.
Our current knowledge of the structure and biochemistry of the LpxC enzymes is limited to very few species. The relatively high degree of primary sequence similarity between the E. coli and P. aeruginosa enzymes (Fig. 2) demonstrates the inadequacy of primary sequences for accurate prediction of functional similarity between proteins. Subtle differences, such as the two nonconserved histidines in the E. coli sequence, may have significant consequences in the overall fold and binding properties. The unexpected differences between the two species' LpxC enzymes highlight the importance of protein structure in drug design. Nuclear magnetic resonance (6) and X-ray (37) structures of LpxC from Aquifex aeolicus have been determined, and the zinc sites of the A. aeolicus and P. aeruginosa enzymes have been studied using extended X-ray absorption fine-structure spectroscopy (21). Structural studies of LpxC from species in addition to A. aeolicus may provide the structural rationale for the differential susceptibilities of these species to a given inhibitor and thereby facilitate the design of inhibitors with much broader specificity than has previously been possible.
Our data suggest that strategies to identify broad-spectrum LpxC inhibitors could be challenging and must take into account the structural differences in LpxC enzymes from different gram-negative bacterial species. The finding that nonenzyme components of cell extracts affect the activity of inhibitors within the bacterial cell adds an additional level of complexity. Target-based antibiotic discovery will be most successful when it is possible to evaluate separately each of the factors that contribute to bacterial growth inhibition: penetration of the cell wall, resistance to efflux, inactivation or sequestration by intracytoplasmic components, and potency toward the target. The use of multiple molecular and biochemical approaches allowed us to evaluate each of these for the LpxC inhibitor L-161,240 and to show that in this case, potency toward the target was the primary factor limiting activity for P. aeruginosa. This conclusion was critical to the discovery of potent small-molecule LpxC inhibitors with antibacterial activity toward P. aeruginosa and other gram-negative pathogens, the details of which have been presented elsewhere (1, 16, 19).
Acknowledgments
This work was supported by PathoGenesis Corporation, Seattle, WA (later merged with Chiron Corporation). C.R.H.R. was supported by NIH grant GM-51310.
REFERENCES
- 1.Andersen, N., J. Bowman, A. Erwin, E. Harwood, T. Kline, K. Mdluli, S. Ng, K. Pfister, and R. Shawar. November2004. Antibacterial agents. Patent application WO 2004/062601 A2.
- 2.Angus, B. L., A. M. Carey, D. A. Caron, A. M. B. Kropinski, and R. E. W. Hancock. 1982. Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild-type with an antibiotic-supersusceptible mutant. Antimicrob. Agents Chemother. 21:299-309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Beall, B., and J. Lutkenhaus. 1987. Sequence analysis, transcriptional organization, and insertional mutagenesis of the envA gene of Escherichia coli. J. Bacteriol. 169:5408-5415. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Chen, M. H., M. G. Steiner, S. E. de Laszlo, A. A. Patchett, M. S. Anderson, S. A. Hyland, H. R. Onishi, L. L. Silver, and C. R. H. Raetz. 1999. Carbohydroxamido-oxazolidines: antibacterial agents that target lipid A biosynthesis. Bioorg. Med. Chem. Lett. 9:313-318. [DOI] [PubMed] [Google Scholar]
- 5.Clements, J. M., F. Coignard, I. Johnson, S. Chandler, S. Palan, A. Waller, J. Wijkmans, and M. G. Hunter. 2002. Antibacterial activities and characterization of novel inhibitors of LpxC. Antimicrob. Agents Chemother. 46:1793-1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Coggins, B. E., X. Li, A. L. McClerren, O. Hindsgaul, C. R. H. Raetz, and P. Zhou. 2003. Structure of the LpxC deacetylase with a bound substrate-analog inhibitor. Nat. Struct. Biol. 10:645-651. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Coggins, B. E., A. L. McClerren, L. Jiang, X. Li, J. Rudolph, O. Hindsgaul, C. R. Raetz, and P. Zhou. 2005. Refined solution structure of the LpxC-TU-514 complex and pKa analysis of an active site histidine: insights into the mechanism and inhibitor design. Biochemistry 44:1114-1126. [DOI] [PubMed] [Google Scholar]
- 8.Guzman, L. M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177:4121-4130. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Hickey, M. J., T. M. Arain, R. M. Shawar, D. J. Humble, M. H. Langhorne, J. N. Morgenroth, and C. K. Stover. 1996. Luciferase in vivo expression technology: use of recombinant mycobacterial reporter strains to evaluate antimycobacterial activity in mice. Antimicrob. Agents Chemother. 40:400-407. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Holloway, B. W. 1955. Genetic recombination in Pseudomonas aeruginosa. J. Gen. Microbiol. 13:572-581. [DOI] [PubMed] [Google Scholar]
- 11.Hyland, S. A., S. S. Eveland, and M. S. Anderson. 1997. Cloning, expression, and purification of UDP-3-O-acyl-GlcNAc deacetylase from Pseudomonas aeruginosa: a metalloamidase of the lipid A biosynthesis pathway. J. Bacteriol. 179:2029-2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Jackman, J. E., C. A. Fierke, L. N. Tumey, M. Pirrung, T. Uchiyama, S. H. Tahir, O. Hindsgaul, and C. R. H. Raetz. 2000. Antibacterial agents that target lipid A biosynthesis in gram-negative bacteria. Inhibition of diverse UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylases by substrate analogs containing zinc binding motifs. J. Biol. Chem. 275:11002-11009. [DOI] [PubMed] [Google Scholar]
- 13.Jackman, J. E., C. R. H. Raetz, and C. A. Fierke. 1999. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase of Escherichia coli is a zinc metalloenzyme. Biochemistry 38:1902-1911. [DOI] [PubMed] [Google Scholar]
- 14.Jackman, J. E., C. R. H. Raetz, and C. A. Fierke. 2001. Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC). Identification of the zinc binding site. Biochemistry 40:514-523. [DOI] [PubMed] [Google Scholar]
- 15.Jacobs, M. A., A. Alwood, I. Thaipisuttikul, D. Spencer, E. Haugen, S. Ernst, O. Will, R. Kaul, C. Raymond, R. Levy, L. Chun-Rong, D. Guenthner, D. Bovee, M. V. Olson, and C. Manoil. 2003. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 100:14339-14344. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Kline, T., N. H. Andersen, E. A. Harwood, J. Bowman, A. Malanda, S. Endsley, A. L. Erwin, M. Doyle, S. Fong, A. L. Harris, B. Mendelsohn, K. Mdluli, C. R. H. Raetz, C. K. Stover, P. R. Witte, A. Yabannavar, and S. Zhu. 2002. Potent, novel in vitro inhibitors of the Pseudomonas aeruginosa deacetylase LpxC. J. Med. Chem. 45:3112-3129. [DOI] [PubMed] [Google Scholar]
- 17.Lee, N. 1980. Molecular aspects of ara regulation, p. 389-410. In J. H. Miller and W. S. Reznikoff (ed.), The operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
- 18.Li, X. Z., D. M. Livermore, and H. Nikaido. 1994. Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin. Antimicrob. Agents Chemother. 38:1732-1741. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.McClerren, A. L., S. Endsley, J. L. Bowman, N. H. Andersen, Z. Guan, J. Rudolph, and C. R. Raetz. 2005. A slow, tight-binding inhibitor of the zinc-dependent deacetylase LpxC of lipid A biosynthesis with antibiotic activity comparable to ciprofloxacin. Biochemistry 44:16574-16583. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.McClerren, A. L., P. Zhou, Z. Guan, C. R. Raetz, and J. Rudolph. 2005. Kinetic analysis of the zinc-dependent deacetylase in the lipid A biosynthetic pathway. Biochemistry 44:1106-1113. [DOI] [PubMed] [Google Scholar]
- 21.McClure, C. P., K. M. Rusche, K. Peariso, J. E. Jackman, C. A. Fierke, and J. E. Penner-Hahn. 2003. EXAFS studies of the zinc sites of UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC). J. Inorg. Biochem. 94:78-85. [DOI] [PubMed] [Google Scholar]
- 22.Miyada, C. G., L. Stoltzfus, and G. Wilcox. 1984. Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression. Proc. Natl. Acad. Sci. USA 81:4120-4124. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Nikaido, H. 1998. The role of outer membrane and efflux pumps in the resistance of Gram-negative bacteria. Can we improve drug access? Drug Resist. Updates 1:93-98. [DOI] [PubMed] [Google Scholar]
- 24.Nikaido, H. 2001. Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria. Semin. Cell Dev. Biol. 12:215-223. [DOI] [PubMed] [Google Scholar]
- 25.Nunn, D., S. Bergman, and S. Lory. 1990. Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili. J. Bacteriol. 172:2911-2919. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Olsen, R. H., G. DeBusscher, and W. R. McCombie. 1982. Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome. J. Bacteriol. 150:60-69. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Onishi, H. R., B. A. Pelak, L. S. Gerckens, L. L. Silver, F. M. Kahan, M. H. Chen, A. A. Patchett, S. M. Galloway, S. A. Hyland, M. S. Anderson, and C. R. H. Raetz. 1996. Antibacterial agents that inhibit lipid A biosynthesis. Science 274:980-982. [DOI] [PubMed] [Google Scholar]
- 28.Pirrung, M. C., L. N. Tumey, A. L. McClerren, and C. R. H. Raetz. 2003. High-throughput catch-and-release synthesis of oxazoline hydroxamates. Structure-activity relationships in novel inhibitors of Escherichia coli LpxC: in vitro enzyme inhibition and antibacterial properties. J. Am. Chem. Soc. 125:1575-1586. [DOI] [PubMed] [Google Scholar]
- 29.Pirrung, M. C., L. N. Tumey, C. R. H. Raetz, J. E. Jackman, K. Snehalatha, A. L. McClerren, C. A. Fierke, S. L. Gantt, and K. M. Rusche. 2002. Inhibition of the antibacterial target UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC): isoxazoline zinc amidase inhibitors bearing diverse metal binding groups. J. Med. Chem. 45:4359-4370. [DOI] [PubMed] [Google Scholar]
- 30.Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
- 31.Schleif, R. 1992. DNA looping. Annu. Rev. Biochem. 61:199-223. [DOI] [PubMed] [Google Scholar]
- 32.Schweizer, H. P. 1998. Intrinsic resistance to inhibitors of fatty acid biosynthesis in Pseudomonas aeruginosa is due to efflux: application of a novel technique for generation of unmarked chromosomal mutations for the study of efflux systems. Antimicrob. Agents Chemother. 42:394-398. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Schweizer, H. P., and T. T. Hoang. 1995. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15-22. [DOI] [PubMed] [Google Scholar]
- 34.Sorensen, P. G., J. Lutkenhaus, K. Young, S. S. Eveland, M. S. Anderson, and C. R. H. Raetz. 1996. Regulation of UDP-3-O-[R-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase in Escherichia coli. The second enzymatic step of lipid A biosynthesis. J. Biol. Chem. 271:25898-25905. [DOI] [PubMed] [Google Scholar]
- 35.Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959-964. [DOI] [PubMed] [Google Scholar]
- 36.Studier, F. W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185:60-89. [DOI] [PubMed] [Google Scholar]
- 37.Whittington, D. A., K. M. Rusche, H. Shin, C. A. Fierke, and D. W. Christianson. 2003. Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis. Proc. Natl. Acad. Sci. USA 100:8146-8150. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Young, K., L. L. Silver, D. Bramhill, P. Cameron, S. S. Eveland, C. R. H. Raetz, S. A. Hyland, and M. S. Anderson. 1995. The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase. J. Biol. Chem. 270:30384-30391. [DOI] [PubMed] [Google Scholar]
- 39.Zimmermann, W. 1980. Penetration of beta-lactam antibiotics into their target enzymes in Pseudomonas aeruginosa: comparison of a highly sensitive mutant with its parent strain. Antimicrob. Agents Chemother. 18:94-100. [DOI] [PMC free article] [PubMed] [Google Scholar]