Interplay of Klebsiella pneumoniae fabZ and lpxC Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis

Emergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation of K. pneumoniae resistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process in K. pneumoniae and how it may be linked to novel biosynthetic pathway inhibitors.

active efflux, overexpression or alteration of the target protein LpxC (35), or mutations in fatty acid biosynthetic genes (fabG in P. aeruginosa or fabZ in E. coli and K. pneumoniae) (15,(35)(36)(37)(38)(39). E. coli fabZ mutations were shown to encode FabZ proteins with reduced activity (37,40), which has been proposed to elevate the distribution of R-3-hydroxyacyl-ACP substrates into the lipid A biosynthetic pathway ( Fig. 1) which ultimately competes with the LpxC inhibitor compound for binding to LpxC (36). Studies of E. coli fabZ mutants in the absence of exposure to an LpxC inhibitor showed that the cells reduce cellular levels of LpxC to balance fatty acid and lipid A synthesis in order to restore membrane homeostasis (37,40). Since a key control point of this process was LpxC, it stands to reason that continuous chemical inhibition of LpxC might select mutants where normal biological control of lipid A biosynthesis at the LpxC step was lost. Without LpxC inhibitor, normal membrane homeostasis would be lost, leading to inhibitor-dependent growth. The availability of potent LpxC inhibitors allowed for selection of mutants in the important Gram-negative pathogen K. pneumoniae by serial passaging (14) that, in addition to becoming less susceptible to the inhibitor, indeed became dependent on the inhibitor for growth. This resulted from a combination of fabZ and lpxC mutations, with the latter appearing to strongly increase cellular levels of altered LpxC protein. The resulting uncontrolled production of lipid A led to an inability to grow unless LpxC was chemically inhibited. The implications of this are discussed.

RESULTS
K. pneumoniae LpxC mutants adapted to grow only in the presence of an LpxC inhibitor emerge during serial passaging. Consistent with previous reports (15,36,37), single-step mutant selection using various LpxC inhibitors consistently yielded fabZ mutants (e.g., strains JWM0009 encoding FabZ R121H , JWM0010 encoding FabZ A69V , and JWM0011 encoding FabZ R126C ) and occasionally selected mutations in lpxC (JWM0107 encoding LpxC V37A ) ( Fig. 2A and B). The mutants did not exhibit noticeable growth defects on common media (e.g., Mueller-Hinton agar [MHA] or LB agar) lacking the LpxC inhibitor. We surmised that more complex phenomena relating to both reduced susceptibility and pathway interactions would be revealed by a serial passaging approach in the presence of compound 2 to allow for the accumulation of mutations and adaptation to continuous exposure to an LpxC inhibitor. Two mutants recovered from that approach had substantially decreased susceptibility to compound 2 (MIC shifted from 2 to Ͼ128 g/ml). Intriguingly, although resistant to compound 2, these mutants failed to grow in the absence of compound 2 (strains JWM0012 and JWM0013 [ Fig. 2A and B]). Additional LpxC inhibitors such as CHIR-090 (31) also induced growth of strains JWM0012 and JWM0013 (data not shown); therefore, the effect was not specific to the compound used for selection.
LpxC inhibitor-dependent growth of K. pneumoniae mutants JWM0012 and JWM0013 is mediated by the combined effect of mutations in fabZ and lpxC. Whole-genome sequencing of K. pneumoniae JWM0012 and JWM0013 revealed the presence of mutations in lpxC and fabZ (encoding LpxC V37G /FabZ R121L and LpxC V37G / FabZ F51L , respectively) along with additional mutations (see Table S1 in the supplemental material). Since lpxC and fabZ mutations were in common between these two mutants, these were both engineered into the susceptible parent K. pneumoniae ATCC 43816 strain background using recombineering. Mutant JWK0148 encoding only the LpxC V37G mutation showed decreased susceptibility to compound 2, indicating that this LpxC variant is itself a determinant of in vitro resistance to an LpxC inhibitor (Fig. 2C). JWK0148 growth was not dependent on compound 2. To our knowledge, this and the LpxC V37A variant of JWM0107 isolated via single-step selection are the first descriptions of LpxC target alterations reducing susceptibility to an LpxC inhibitor in K. pneumoniae.
Mutants JWK0150 and JWK0151 were engineered to encode LpxC V37G plus FabZ R121L or FabZ F51L, respectively. Each of these engineered mutants required compound 2 in order to grow (Fig. 2C). It should be noted that the originally selected mutants required somewhat higher levels of compound 2 to induce growth than the  FabZR121H, JWM0010 encoding FabZA69V,  JWM0011 encoding FabZR126C), an lpxC mutant (JWM0107 encoding LpxCV37A) or fabZ/lpxC double mutants (JWM0012 encoding LpxCV37G/FabZR121L and JWM0013 encoding LpxCV37G/FabZF51L) were assessed for growth in the presence of compound 2 using broth medium. Individual fabZ or lpxC mutations decreased susceptibility to compound 2 by about four-to eightfold as expected. Both mutations together further enhanced resistance and led to compound 2-dependent growth. (C) The roles of lpxC mutation and the combined lpxC/fabZ mutations were confirmed using engineered K. pneumoniae ATCC 43816 derivatives (JWK0148 encoding LpxCV37G, JWK0150 encoding LpxCV37G/FabZR121L, and JWK0151 encoding LpxCV37G/FabZF51L). engineered mutants and were ultimately even less susceptible to the inhibitor (Fig. 2C). This likely resulted from the presence of additional mutations in the passaged mutants contributing to decreasing susceptibility to compound 2. Indeed, isolated mutant JWM0012 has an additional mutation inactivating RamR (Table S1). RamR is the transcriptional repressor of RamA, which is a transcriptional activator of efflux pumps AcrAB (41). Recently, RamA was also reported to activate transcription of genes in the lipid A biosynthesis pathway, including lpxC (42).
LpxC V37G and LpxC G36R accumulate to high levels in the cell. We mapped the selected LpxC mutations onto an available E. coli LpxC inhibitor costructure. K. pneumoniae LpxC V37G, as well as LpxC G36R and LpxC V37G variants that we had selected for reduced susceptibility to the LpxC inhibitor in E. coli (Table S1), localized at a distance from the binding pocket (see Fig. S1 in the supplemental material). A previously described E. coli variant LpxC I38T , which decreases susceptibility to an LpxC inhibitor BB-78484 (36) that is structurally different from compound 2, also localized to this region of the protein. These results suggested that this region of LpxC is important for determining susceptibility to a range of different LpxC inhibitors.
Since these mutations did not appear to affect the LpxC inhibitor binding site, we hypothesized that they might act by somehow mediating increased LpxC protein levels. Increased abundance of LpxC would be consistent with the ability of these mutations to reduce susceptibility to LpxC inhibitors in general. Western blot analysis showed a clear increase in LpxC abundance relative to the parent strain in K. pneumoniae encoding the LpxC variant LpxC V37G (strain JWM0148) and perhaps even larger accumulation in mutants encoding FabZ R121L /LpxC V37G (JWM0012) and FabZ R121 /LpxC V37G (JWM0150) (Fig. 3). There was a modest (threefold) increased abundance of lpxC transcripts in mutants JWK0148 and JWK0150 (data not shown), which presumably contributes to the increased level of LpxC, but the increase of protein abundance is much higher than threefold. This suggests that LpxC V37G may be more stable than wild-type LpxC. Since the V37G substitution is not localized in the compound binding site and may not significantly impact compound 2 binding, increased abundance of LpxC V37G may decrease susceptibility to compound 2 via a compound titration effect.
FabZ R121L and LpxC V37G together lead to loss of control of the lipid A biosynthetic pathway and severe overproduction of LPS. Synthesis of LPS needs to be tightly controlled and balanced with phospholipid (inner membrane) biosynthesis (37,38). It was recently shown that E. coli responds to the fatty acid pathway defect  by fabZ mutations by reducing cellular levels of LpxC, presumably decreasing the production of lipid A (37,43,44). However, as shown above, LpxC V37G accumulated to levels higher than wild-type levels within cells regardless of the presence or absence of a fabZ mutation, and indeed may have accumulated slightly higher levels in the presence of fabZ mutations ( Fig. 3A and B). Assuming that wild-type K. pneumoniae exhibits similar coregulation of FabZ and LpxC to that described for E. coli, our observations suggest that LpxC V37G interferes with the normal ability to control LpxC levels in response to defects in FabZ. Consistent with this, JWM0012 (FabZ R121L / LpxC V37G ) cells exhibited an extreme accumulation of LPS within the periplasm when subcultured into medium lacking compound 2 ( Fig. 4 and Fig. S2) as would be expected from such dysregulation. Similar results were seen for the engineered fabZ/lpxC mutant JWK0150 (Fig. 4). Interestingly, there were no obvious membrane defects in cells expressing only LpxC V37G alone (Fig. 4C), indicating that increased LpxC levels in this mutant, in the absence of additional substrate redirection resulting from defective FabZ, were insufficient to cause serious LPS accumulations. The extreme level of LPS (especially relative to inner membrane) in the fabZ/lpxC mutant indicated an inability to establish or maintain membrane homeostasis in these mutants in the absence of compound 2. Compound 2 at 32 g/ml prevented LPS accumulation (Fig. 4), and , and the passaged fabZ/lpxC double mutant (JWM0012) (E) as examined by transmission electron microscopy. Compound 2-dependent mutants JWK0150 and JWM0012 exhibited a severe accumulation of membranous material (arrows) when subcultured from medium with compound 2 to medium without compound 2 (left panels). This did not occur in cells subcultured into compound 2-containing medium (right panels). The scale is indicated in each image. exposure to compound 2 also allowed these mutants to grow on MacConkey agar plates that contain bile salts which prevent the growth of Enterobacteriaceae if they have membrane permeability defects (data not shown). Moreover, compound 2 at sublethal levels strongly sensitized wild type, but not the fabZ/lpxC mutant K. pneumoniae to rifampin. Therefore, inhibition of LpxC induced an outer membrane permeability defect in wild-type cells, whereas it prevented a membrane defect in the mutants (Fig. S3). The apparent lack of membrane defects in the mutants treated with compound 2 provides further evidence that membrane homeostasis has been restored in the fabZ/lpxC mutants via chemical inhibition of LpxC.
Overproduction of lipid A leads to accumulation of lipid A pathway intermediates. The periplasmic accumulation of LPS seen here for our fabZ/lpxC mutants is reminiscent of that observed in cells with defects in the Lpt LPS transport machinery (45,46) or in cells treated with the LptD inhibitor POL7080 (47). Therefore, the rate of lipid A (LPS) synthesis was so high as to seemingly exceed the capacity of LPS transporters (e.g., LptD) to translocate and assemble the LPS into a properly formed outer membrane. Inactivating lptD in A. baumannii did not result in periplasmic accumulation but did lead to toxic accumulation (backing up) of detergent-like lipid A pathway intermediates that interfered with growth, and similar to what we see here, growth was improved by chemical inhibition of LpxC (48). Therefore, we determined whether there might also be accumulation of lipid A pathway intermediates occurring in our K. pneumoniae fabZ/lpxC mutants. Analysis by LC-MS/MS showed that K. pneumoniae mutants harboring only a fabZ mutation accumulated a significant amount of LpxA product (Fig. 5). This would be consistent with redirection of substrate from fatty acid into the lipid A pathway and/or the compensatory downregulation of LpxC in fabZ mutants (36,37,40). The accumulation of LpxA product (LpxC substrate) measured here may however be consistent with the notion of increased abundance of substrate competing for binding to LpxC as a mechanism of resistance to LpxC inhibitors (36). To assess intermediate accumulation in the fabZ/lpxC mutant strain JWM0012, cells were cultivated initially in the presence of compound 2 and then dispensed into 96-well plates with twofold dilutions of compound 2, including wells containing no compound. Growth was monitored by optical density at 600 nm (OD 600 ) , and cells were harvested for LC-MS/MS analysis (Fig. 6). Cells subcultured without sufficient compound 2 showed clear accumulation of lipid X and disaccharide 1-monophosphate (DSMP) over time. This accumulation progressively decreased as cells were subcultured in increasing concentrations of compound 2. In the presence of higher concentrations of compound 2 (circa 4 to 8 g/ml), lipid X and DSMP levels were similar to those of the wild-type strain. These results raise the possibility that toxic accumulation of lipid A pathway intermediates at the inner membrane might contribute to the inability of these mutants to grow without inhibition of LpxC.
K. pneumoniae fabZ/lpxC mutants escape from inhibitor-dependent growth at high frequency. The lpxC/fabZ mutations harbored by strain JWM0012 confer an inability to grow in the absence of an LpxC inhibitor. To examine the ability to escape from this extreme growth defect, cells were plated for mutant selection on solid medium lacking LpxC inhibitor. Mutants arose at a frequency of circa 6 ϫ 10 Ϫ8 . A range of phenotypes were observed across several mutants picked for further study, indicating that different mechanisms of escape could be selected. Some mutants reverted to wild-type susceptibility to compound 2 (strains JRW0037 and JRW0039 [ Table S1]), while others continued to demonstrate reduced susceptibility to compound 2 but did not require it for growth (JRW0040, JRW0041, and JRW0042 [ Table S1]). Still others grew in the absence of compound 2 and in the presence of a high concentration of compound 2, but not with intermediate concentrations, similar to the eagle phenotype reported earlier (15) (JRW0035, JRW0036, and JRW0038 [ Table S1]). Genome sequencing revealed that several of these mutants had acquired suppressor mutations in lpxC encoding the following amino acid substitutions in addition to the original V37L substitution: JRW0037 (L208P), JRW0038 (T6S), JRW0039 (P102L), and JRW0040 (T6S and D156Y) (Table S1). Western blot analysis of these mutants showed that the abundance of LpxC protein was still elevated relative to strains expressing wild-type LpxC (data not shown). This suggested that these changes, rather than restoring normal LpxC levels, functioned to reduce the catalytic efficiency of LpxC, effectively producing the same result as would a reduction in LpxC levels (or chemical inhibition); however, this remains to be confirmed. One mutant (JRW0041 [Table S1]) harbored a mutation in lpxA (encoding G201R) (Table S1). Although this change is not localized within the catalytic domain of LpxA based on the available published P. aeruginosa and E. coli LpxA structure (not shown), we suspect that this mutation somehow reduces LpxA activity, thereby reducing the flow of substrate into LpxC and allowing compound-independent growth. Intriguingly, this mutant had exceptionally high levels of LpxC protein (data not shown), suggesting a possible regulatory response to the lack of LpxC substrate being produced in this mutant. Last, we identified a mutation inactivating the heat shock stress response protein DnaK in one of the mutants (JRW0035 [Table S1]). The mechanism by which this mutation would restore compound 2-independent growth is not known; however, DnaK has been shown to be important in a variety of processes affecting survival under oxidative stress, nutrient limitation, and antibiotic exposure (49).

DISCUSSION
Although rare, cases where bacteria have developed a dependence on an antibacterial compound for growth have been described, for example with streptomycin, colistin, and vancomycin (8,(50)(51)(52)(53)(54)(55). Here we describe the isolation of K. pneumoniae mutants whose growth requires the inhibition of LpxC. Consistent with previous reports with other LpxC inhibitors (15,36), individual mutations in the fatty acid biosynthetic gene fabZ and in the gene that encodes the target protein, lpxC, decreased susceptibility of K. pneumoniae to the LpxC inhibitor compound 2. However, K. pneumoniae mutants that encoded LpxC V37G together with either FabZ R121L or FabZ F51L failed to grow in the absence of the LpxC inhibitor.
The LpxC variants identified here accumulated to much higher than normal levels in the mutant cells, possibly due to an apparent threefold increase of lpxC transcript abundance but possibly also due to increased protein stability. Overexpression of LpxC was a potential contributor to reducing compound susceptibility by a straightforward titration mechanism, as has been reported previously for P. aeruginosa (35). However, the increased levels of LpxC in conjunction with the fabZ mutations were of interest, as they prevented cell growth. Synthesis of LPS needs to be balanced with phospholipid (inner membrane) biosynthesis for formation of a functional cell envelope (37)(38)(39). Single fabZ mutations selected on LpxC inhibitors in E. coli may cause redirection of substrate from fatty acid synthesis into lipid A synthesis or simply decrease phospholipid synthesis, either of which might be expected to disturb this balance. It was recently shown that E. coli responds to fabZ mutations by reducing cellular levels of LpxC, presumably decreasing production of lipid A (37,43,44). However, the accumulation of LpxC V37G in our mutants at higher than wild-type levels regardless of the presence or absence of a fabZ mutation suggested that the normal ability to adequately control LpxC levels had been lost. Presumably, this could occur during passaging in compound 2 because normal regulation was replaced by progressively increasing chemical inhibition of LpxC. The overly robust production of lipid A and severe accumulation of LPS in cells in the absence of the LpxC inhibitor represent an inability of these mutants to properly maintain membrane homeostasis and extend our understanding of the importance of this process to K. pneumoniae.
In addition to the overall membrane imbalance occurring in our mutants, there was an accumulation of lipid A pathway intermediates. Certain intermediates of the lipid A biosynthetic pathway (e.g., the substrate of LpxD) are detergent-like compounds, and it was suggested that if they should accumulate in cells, they would be toxic (56,57). The idea of toxic accumulation has perhaps been best exemplified for biosynthetic pathways whose end products are not required for growth. An example is the biosynthesis of teichoic acid in Gram-positive bacteria. Genetic deletion of enzymes occurring early in the pathway is tolerated, but deletion of later steps is not, the so-called "essential gene paradox" (58). This has been attributed to toxic accumulation of pathway intermediates, although other explanations are possible. An essential gene paradox for lipid A biosynthesis was recently reported whereby LpxH and LpxK, which occur later in the biosynthetic pathway, were essential for in vitro growth of A. baumannii ATCC 19606 even though the earlier catalytic enzymes LpxA or LpxC were dispensable in that strain background (i.e., lipid A synthesis is not essential) (59,60). Similar to our observations here, growth of A. baumannii ATCC 19606 downregulated for LpxH or LpxK was restored by an LpxC inhibitor (LpxC inhibitor-dependent growth), and this was associated with reduction of toxic intermediate accumulation. Deletion of the outer membrane LPS (referred to as lipooligosaccharide [LOS] in A. baumannii) transporter gene lptD in this strain reduced but did not eliminate cell-associated LOS and caused growth and permeability defects in cells, consistent with toxic accumulation, and these defects were relieved by inhibition of LpxC (48). The association of lipid A intermediates with growth inhibition described here may provide the first glimpse of a contribution of toxic intermediates to growth inhibition in K. pneumoniae, where lipid A synthesis is essential. This also provides further support to the notion that enzymes later in the lipid A pathway, such as LpxK, could have an added advantage of generating this type of toxic accumulation if inhibited and therefore be desirable therapeutic targets (44).
We do not know precisely how control of the cellular level of the LpxC variant selected here becomes dysregulated. One mechanism by which LpxC levels are controlled inside cells is via proteolytic degradation by the essential protease FtsH (61)(62)(63)(64). An FtsH recognition motif was identified in the C terminus of LpxC (61,62), and an additional site close to the LpxC N terminus may also be involved (62). A temperaturesensitive ftsH mutant accumulated LpxC upon shift to the nonpermissive temperature (65). More recently, it was proposed that a second protease may also play a role in controlling LpxC levels in E. coli (43). Since accumulation of LpxC in our mutants is mediated by amino acid substitutions located near the N terminus, one possibility is that these substitutions render LpxC refractory to proteolytic cleavage, but this remains to be experimentally shown. Accumulation of LpxC in the temperature-sensitive E. coli ftsH mutant also led to severe overproduction of membrane in the periplasm (65), similar to what we observed here for LpxC inhibitor-dependent mutants subcultured without compound 2. This suggests that accumulated LpxC alone can promote a significant overproduction of lipid A even in the absence of a mutation in fabZ, although the LpxC V37l variant studied here did not show significant envelope abnormalities. The resulting membrane imbalance would be expected to affect the membrane permeability barrier and/or cause a fitness deficit. Consistent with this, K. pneumoniae strains harboring mutations only in lpxC (JWK0148-LpxC V37G and JWM0107-LpxC V37A ) grew well under our typical static experimental growth conditions but grew more slowly under shaking conditions, and growth was improved in the presence of compound 2. Growth was also poor on MacConkey agar, and sensitivity to rifampin was higher than for the wild-type strain, consistent with membrane defects (data not shown). This indicated LpxC V37G or LpxC V37A alone resulted in membrane imbalance, but not as severely as when combined with a fabZ mutation.
In conclusion, we exploited the availability of potent LpxC inhibitors to enable a K. pneumoniae serial passaging approach linking the selection of resistance to the generation of inhibitor-dependent growth. Individual fabZ mutations or mutations in the target gene lpxC decreased susceptibility to compound 2 as expected; however, the combination of these led to inhibitor-dependent growth. Underlying this was the idea that chemical inhibition of LpxC allowed mutants to emerge that had lost their biological mechanism(s) necessary to establish and maintain membrane homeostasis. This provides further support for the importance of the proper balancing of membrane synthesis and extends our understanding of this process to the important human pathogen K. pneumoniae. We also provide an intriguing view into the possibility of a contribution of toxic intermediate accumulation to growth inhibition in the context of an essential pathway, which may inform the selection of therapeutic targets within this pathway. Last, it is not known whether these mutants could emerge in the clinic during treatment with an LpxC inhibitor, but it is tempting to speculate that should this occur, the mutants may not be able to survive upon withdrawal of the drug.

MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table S1 in the supplemental material. K. pneumoniae ATCC 43816 and E. coli ATCC 25922 were purchased from the American Type Culture Collection (ATCC). Cells were routinely grown in cation-adjusted Mueller-Hinton broth (MHB) (3.0 g/liter beef extract, 17.5 g/liter acid hydrolysate of casein, 1.5 g/liter starch, 20 to 25 mg/liter calcium, 10 to 12.5 mg/liter magnesium) or lysogeny broth (LB) (10 g/liter tryptone, 5 g/liter yeast extract, and 10 g/liter NaCl). Media were supplemented with carbenicillin (500 g/ml for K. pneumoniae) for mutant construction by recombineering (66). The LpxC inhibitor, compound 2 (14), was synthesized at Novartis.
Determination of MICs and serial passage for isolating mutants resistant to LpxC inhibitor compound 2. For MIC determinations, bacterial strains were streaked on MHB agar or MHB agar supplemented with compound 2 as indicated and grown overnight. LpxC inhibitor compound 2 was twofold serially diluted in the 96-well round-bottom plate in 50 l of MHB or LB. Bacterial suspensions were prepared from overnight colonies using BBL prompt (BD Biosciences, San Jose, CA) according to the supplied instructions and used to introduce approximately 5ϫ 10 5 colony-forming units (CFU) in 50 l in each well. Plates were incubated at 37°C, and bacterial growth was determined by monitoring optical density at 600 nm (OD 600 ).
Serial passage experiments were based on the 96-well plate MIC format described above. From the MIC determination, 2 l of culture (50ϫ dilution) from wells at one compound 2 dilution step below the MIC (i.e., the highest concentration of compound 2 where the cells grew) was used to inoculate a second plate set up similarly to the first plate. This process was repeated for a total of 12 passages, and the MIC of the culture progressively increased. Culture from a passage that starts to show compound 2-dependent growth was streaked on MHB agar containing compound 2 to isolate individual mutants. Individual mutants were then tested with the MIC assay again to ensure they retain the growthdependent phenotype.
Whole-genome sequencing of isolated mutants. Genomic DNA was prepared from bacterial cells using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Redwood City, CA) according to the manufacturer's instructions, except that samples were eluted in and then diluted to 8 ng/l with nuclease-free water. Whole-genome sequencing and identification of mutations were performed according to standard protocols.
Construction of mutants JWK0148 (LpxC V37G ), JWK0150 (LpxC V37G /FabZ R121L ), and JWK0151 (LpxC V37G /FabZ F51L ) in K. pneumoniae ATCC 43816. Plasmids and oligonucleotides used in this study are listed in Tables S1 and S2. K. pneumoniae ATCC 43816 was transformed with plasmid pTU430 (Table S1), which harbors the recombinase from pKD46 (66), for chromosomal engineering and the sacB gene for counterselection. DNA substrates encoding amino acid substitutions for LpxC V37G , FabZ R121L , and FabZ F51L were PCR amplified from mutant strain JWM0012 or JWM0013 using primer pairs Kp.lpxCF/ Kp.lpxCR or Kp.fabZF/Kp.fabZR and gel purified. K. pneumoniae ATCC 43816 harboring the recombinase plasmid pTU430 was grown overnight in LB with carbenicillin (500 g/ml) and diluted 1:100 in fresh LB with carbenicillin (150 g/ml) and 0.2% arabinose and grown to an OD 600 of ϳ0.6. Cells were pelleted by centrifugation and washed twice in ice cold 10% glycerol. The linear DNA substrates encoding mutations were then introduced to cells by electroporation. Transformants were plated on LB plates with compound 2 at 4 g/ml. Candidate mutants were cured of plasmid pTU430 by plating on LB agar with 8% sucrose and lacking NaCl. A PCR-confirmed mutant harboring the chromosomal mutation encoding LpxC G37V was designated JWK0148. Mutations encoding FabZ R121L and FabZ F51L were then introduced into the genome of strain JWK0148 by a second round of recombineering. PCR-confirmed mutants designated JWK0150 and JWK0151 had mutations encoding LpxC V37G /FabZ R121L and LpxC V37G /FabZ F51L , respectively.
Western blot for detection of LpxC proteins. Bacterial strains were grown overnight in 5 ml LB (K. pneumoniae ATCC 43816 and lpxC mutant JWK0148) or LB supplemented with 32 g/ml compound (fabZ/lpxC mutants JWM0012 and JWK0150) at 37°C with shaking. The cultures were diluted 1:20 the next day in 30 ml LB alone or supplemented with 32 g/ml compound 2 and incubated again in the 37°C shaker. In either the presence or absence of compound 2, lpxC or fabZ/lpxC mutants showed a significantly higher LpxC abundance compared to the wild type. For simplification, we showed only the results for strains grown with 32 g/ml compound 2, in which wild-type culture was significantly suppressed. (In comparison, when grown in the absence of compound 2, growth of fabZ/lpxC mutants JWM0012 or JWK0150 was significantly suppressed.) Culture density was monitored by OD 600 every half hour, and cells were collected at 2 h. The culture was normalized to an OD 600 of 0.5, pelleted, and suspended in 1:2 Tricine sample buffer, sonicated for 1 min, and boiled at 70°C for 20 min. Samples were loaded and run on a Bolt 4 to 12% Bis-Tris Plus gel (catalog no.NW04127BOX; Thermo Fisher Scientific, Waltham, MA) with MES buffer (50 mM MES, 50 mM Tris, 1 mM ETDA, 0.1% SDS). Proteins were then transferred to a nitrocellulose membrane using an iBlot system (Thermo Fisher Scientific, Waltham, MA) according to the supplied instructions. LpxC protein was detected using an iBind Flex Western device (Thermo Fisher Scientific, Waltham, MA) and visualized with the Odyssey CLX imaging system (LI-COR Biosciences, Lincoln, NE).
Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) detection of lipid A pathway intermediates. (i) Sample preparation. K. pneumoniae ATCC 43816, fabZ mutant JWM0009, or fabZ/lpxC JWM0012 was grown overnight in MHB or MHB supplemented with 32 g/ml of compound 2 (for strain JWM0012). The cultures were then diluted to an OD 600 of ϳ0.05 in fresh MHB medium and grown for 1 h in a flask with shaking at 37°C to an OD 600 of ϳ0.15 to 0.2. An 11 point 1:2 serial dilution of compound 2 was made in DMSO at 100ϫ, and 10 l was stamped into 96-well 2-ml deep-well plates for a final assay concentration series starting at 128 g/ml. DMSO was also stamped at a 10-l volume for the no-compound control. One plate was used for each time point, and each serial dilution of the compound was replicated twice. Cells were added to the 96-well plates at a volume of 1 ml, and then plates were sealed with a breathable seal and incubated at 37°C with shaking. After each time point, the plate was removed from the incubator, and 100 l of the cell solution was transferred to a clear 96-well plate for OD 600 reading. To the remaining 900 l of cell culture, 1 ml of Genlantis SoluLyse detergent lysis buffer was added, and the C10 P. aeruginosa LpxA product (Alberta Research Chemicals, Edmonton, Canada) internal standard was spiked in at a final concentration of 1 nM. Plates were shaken at room temperature for 30 min before being spun at 3,000 rpm for 15 min to pellet cellular debris. Solid-phase extraction was performed using Waters Oasis WAX SPE 96-well plates (10 mg sorbent per well, 30-m particle) on a vacuum manifold. Solid-phase extraction products (SPE) were conditioned with 500 l of methanol and reequilibrated with 500 l of water. From the pelleted cell lysate, two 900-l portions of supernatant were transferred to SPE, with flowthrough being discarded. SPE were washed with 500 l of 2% formic acid, followed by 500 l of acetonitrile. LPS intermediates were eluted from SPE with 75 l of 5% ammonium hydroxide in methanol into a 96-well polypropylene plate containing 15 l of 10% formic acid in methanol per well to acidify the samples to approximately pH 5 to 6. Note that allowing the LPS biosynthetic intermediates to incubate in basic solution will result in poor results due to hydrolysis.
(ii) Detection of LPS intermediates by normal-phase LC-MS/MS. Multiple reaction monitoring (MRM) data were acquired on a Sciex 4000 QTRAP mass spectrometer with Turbo V ion source coupled to an Agilent 1100 LC. Both systems are well-suited to normal-phase (NP) operation when the appropriate glass and steel capillaries are used, as well as appropriate pump seals. Other LC-MS systems may be less suitable for the buffers employed, consult the vendor. Mobile phase A consisted of 80% chloroform, 19.5% methanol, and 0.5% ammonium hydroxide. Mobile phase B consisted of 45% chloroform, 45% methanol, 9.5% water, and 0.5% ammonium hydroxide. Flow was applied at 200 l/min to a Waters Acquity UPLC BEH HILIC column (130-Å pore; 1.7-m particle) (2.1 by 100 mm). Samples were kept at 4°C in the autosampler prior to injecting 10 l onto the column. Samples were loaded in 10% mobile phase B, and this composition was held for the first minute of chromatography. Mobile phase B was increased to 70% at 3 min and to 99% at 6 min, held at 99% until 9 min, and then reduced to 10% at 9.1 min. The system was reequilibrated for 4 min between sample injections. MS/MS data were collected between minutes 2 to 11. The mass spectrometer source heater was set at 450°C, IonSpray voltage of Ϫ4,500 V, curtain gas of10 liters/min, collision gas of 8 liters/min, nebulizer gas of 35 liters/min, and heater gas of 40 liters/min. MRM settings are shown in Table S3. MRM data were acquired with a duty cycle of 1.26 s per scan. All transitions were tuned on samples of authentic standards in infusion mode prepared as described previously (59). Integrated retention times were matched to authentic standard elution profiles as well. Peaks were integrated using Skyline software and then normalized to OD 600 and internal standard for each monitored metabolite. For Fig. 5, results are shown for the 8-h time point and are the average of eight replicates. Figure 6 shows the time course experiment, with results from duplicates at the 1-, 3-, 5-, and 8-h time points.