A mutant Escherichia coli that attaches peptidoglycan to lipopolysaccharide and displays cell wall on its surface
A peptidoglycan ( PG ) cell wall is an essential extracytoplasmic feature of most bacteria ( Singer et al . , 1989 ) ; this essentiality has made its biogenesis a fruitful target for antibiotics , including vancomycin and penicillin . The cell wall is directly exposed to the extracellular milieu in Gram-positive bacteria , but is shielded in Escherichia coli and other Gram-negative species by a highly selective permeability barrier formed by the outer membrane ( OM ) . The OM prevents influx of antibiotics , such as vancomycin , restricting their access to intracellular targets ( Eggert et al . , 2001; Ruiz et al . , 2005 ) . Lipopolysaccharide ( LPS ) forms the surface-exposed outer leaflet of the OM and is key to the barrier function ( Osborn et al . , 1972; Kamio and Nikaido , 1976; Nikaido , 2003 ) . LPS is a glycolipid consisting of a ‘lipid A’ anchor within the bilayer , and a set of covalently attached distal ‘core’ saccharides ( Raetz and Whitfield , 2002 ) . LPS is made at the cytosolic leaflet of the inner membrane ( IM ) before being flipped to the periplasmic leaflet ( Zhou et al . , 1998 ) . A transenvelope complex of seven lipopolysaccharide transport proteins ( LptABCDEFG ) delivers LPS from the IM to the OM ( Ruiz et al . , 2009; Chng et al . , 2010a ) . A sub-complex of the β-barrel LptD and lipoprotein LptE resides within the OM and accomplishes the final step of inserting LPS into the outer leaflet ( Chng et al . , 2010b ) . A recently described lptE mutation ( lptE613 ) causes defective LPS transport and leads to increased antibiotic sensitivity ( Malojčić et al . , 2014 ) . To better understand the basis of the lptE613 defect , we isolated spontaneous suppressors that restored antibiotic resistance . One such vancomycin-resistant suppressor mapped to the waaL gene , the product of which is an IM glycosyltransferase that attaches O-antigen ( O-Ag ) oligosaccharides to LPS ( Han et al . , 2012; Ruan et al . , 2012 ) . Indeed , the suppressed strain is certainly no more vancomycin sensitive than is the corresponding wild-type control ( Figure 1A ) . However , this suppressor ( waaL15 herein ) was not specific for lptE613 or even for LPS transport defects . The waaL15 mutation increases vancomycin resistance in strains carrying bamB or bamE null mutations that disrupt the OM barrier by causing defects in β-barrel protein assembly ( Figure 1B ) ( Ricci and Silhavy , 2012 ) . Moreover , waaL15 also increases vancomycin-resistance even in the wild-type strain ( Figure 1A ) . The suppressor does not qualitatively improve the OM barrier , since it did not increase resistance against other antibiotics ( Figure 1B ) . So , waaL15 provides a vancomycin-specific resistance mechanism across different strains . 10 . 7554/eLife . 05334 . 003Figure 1 . A mutant O-antigen ligase increases vancomycin resistance . ( A ) waaL15 provides a strain-independent increase in vancomycin resistance . Isogenic strains , differing by a point mutation in waaL , were plated by serial dilution on LB agar containing indicated amounts of vancomycin . ( B ) waaL15 does not improve resistance against other antibiotics . Antibiotic discs containing either 15 μg erythromycin or 5 μg rifampin were placed on LB agar overlays inoculated with the indicated strains . Diametric zones of growth inhibition were measured across the disc . The disc diameter was 6 mm and this value represents growth at the disc . DOI: http://dx . doi . org/10 . 7554/eLife . 05334 . 003 The domesticated E . coli K-12 does not produce the normal substrate ( O-Ag ) of WaaL ( Liu and Reeves , 1994 ) and a waaL deletion does not suppress vancomycin sensitivity , indicating that waaL15 is a gain-of-function mutation . Thus , the WaaL15 mutant O-Ag ligase , which contains an F332S substitution , must have an altered activity . Silver-staining of isolated LPS confirmed that WaaL15 modifies LPS with additional sugars to produce an additional glycoform ( LPS* ) , detected as a higher molecular-weight band that is absent in waaL+ ( Figure 2A ) . WaaL can use two minor saccharide substrates to modify LPS in E . coli K-12: enterobacterial common-antigen ( ECA ) and colanic acid ( CA ) . ECA-modified LPS is a minor constituent of the OM ( Schmidt et al . , 1976; Meredith et al . , 2007 ) . Production of CA is regulated by the Rcs phospho-relay stress response system , and CA-modified LPS ( called ‘M-LPS’ ) is only detectable during severe envelope stress ( Meredith et al . , 2007 ) . Perhaps waaL15 had improved the utility of one , or both , of these substrates . However , LPS silver-staining revealed that LPS* remained detectable when we inactivated biosynthesis of ECA ( rff ) , CA ( cpsG ) , or both these polysaccharides ( rff cpsG ) ( Figure 2A ) . Moreover , if we increase the amounts of a competing substrate by introducing the rcsC137 mutation to activate expression of the genes for CA biosynthesis ( Gottesman et al . , 1985 ) , we actually observed lowered LPS* abundance at the expense of increased M-LPS ( Figure 2B ) . Notably , the decrease in LPS* correlated with a significant reduction in vancomycin-resistance , providing evidence that LPS* molecules directly mediate the resistance ( Figure 2C ) . Similarly , if O-antigen biosynthesis is restored by introducing a wild-type wbbL gene , we observe lowered LPS* at the expense of wild-type LPS and vancomycin resistance is reduced . We conclude that WaaL15 is able to use a new substrate and thereby generate a previously uncharacterized LPS glycoform that provides a specific mechanism for vancomycin resistance . 10 . 7554/eLife . 05334 . 004Figure 2 . Mutant O-antigen ligase produces a novel form of LPS that is directly responsible for vancomycin resistance . ( A ) WaaL15 uses a novel substrate to produce a new LPS glycoform . Isolated LPS was resolved by SDS-PAGE and detected by silver staining . A higher molecular weight glycoform ( LPS* ) appears in waaL15 strains . Mutations that inactivate biosynthesis of ECA ( rff::Tn10-66 ) or CA ( ΔcpsG::kan ) do not abrogate LPS* production . ( B ) Overproduction of CA leads to decreased LPS* abundance . Isogenic strains were constructed to express either wt rcsC+ or the rcsC137 mutant allele that hyper-activates CA biosynthesis . LPS was isolated and visualized as in ( A ) . LPS molecules modified with a one- or two- CA repeat units are labeled M-LPS and M2-LPS , respectively . ( C ) Reduced LPS* levels correlate with reduced vancomycin resistance . Strains were plated by serial dilution onto LB agar supplemented with vancomycin . DOI: http://dx . doi . org/10 . 7554/eLife . 05334 . 004 All native WaaL substrates contain carbohydrates linked to a common undecaprenyl ( Und ) lipid carrier . PG biosynthesis involves a disaccharide pentapeptide ( DPP ) linked to the same Und carrier , a molecule called lipid II ( Figure 3A ) . To directly determine if lipid II is a substrate for WaaL15 , we treated isolated LPS* with the muralytic enzyme mutanolysin ( Figure 3A , B ) . Digestion of purified LPS* , but not LPS , liberated near-stoichiometric quantities of fragments that were identified by mass spectrometry as DPP or derivatives with a tetrapeptide stem ( Figure 3C ) . Importantly , there is no evidence for cross-linked products suggesting that lipid II was the source of the LPS* glycosylation ( Lebar et al . , 2013 ) . 10 . 7554/eLife . 05334 . 005Figure 3 . Lipid II is the glycosyl donor for LPS* . ( A ) Structure of lipid II and schematic of peptidoglycan cleavage by mutanolysin that releases disaccharide pentapeptide ( ‘A’ ) and tetrapeptide ( ‘B’ ) species . ( B ) Treatment of waaL15 isolated LPS with mutanolysin cleaves the LPS* modification . ( C ) LPS* is glycosylated with equivalent amounts of lipid II-sourced disaccharide pentapeptide and tetrapeptide . Isolated and purified LPS* from waaL15 and LPS from waaL+ were treated with mutanolysin and analyzed by LC-MS . Total ion chromatogram for degradation products ( i and ii ) , and the extracted ion chromatogram for LPS* degradation ( iii ) are shown . M+H and ( M+2H ) /2 ions corresponding to each fragment were extracted ( A: 1013 . 3 + 507 . 2; B: 942 . 3 + 471 . 7 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 05334 . 00510 . 7554/eLife . 05334 . 006Figure 3—figure supplement 1 . The waaL15 mutation does not affect the PG cell wall . ( A ) The total ion chromatograms ( TIC ) of mutanolysin digested PG cell wall from waaL+ strain MG1210 , and waaL15 strains MG1211 are nearly identical . Disaccharide pentapeptide ( DPP , fragment A ) is present in mutanolysin digested LPS* but not in the PG cell wall samples . ( B ) Mass spectra and structures for reduced DPP ( fragment A ) and reduced disaccharide tetrapeptide ( fragment B ) from mutanolysin digested LPS* . DOI: http://dx . doi . org/10 . 7554/eLife . 05334 . 006 There are several carboxypeptidases in the periplasm that remove the terminal D-Alanine ( D-Ala ) from DPP to produce the tetrapeptide derivative . Indeed , E . coli PG contains negligible amounts of pentapeptide stems ( Figure 3—figure supplement 1 ) . Figure 3C shows that about 50% of the LPS* is sequestered before it can be attacked by one of these carboxypeptidases . It seemed likely that sequestration happens because the molecule is transported from the periplasm to the cell surface . Peptide stems from adjacent peptidoglycan strands in the cell wall are cross-linked via transpeptidation between the penultimate D-Ala on one stem and a meso-diaminopimelic acid ( m-DAP ) residue on a nearby stem ( Vollmer et al . , 2008 ) . Extensive cross-linking produces a rigid macromolecular meshwork that is vital to cell wall function . Vancomycin binds and sequesters the terminal D-Ala-D-Ala residues of a pentapeptide stem in order to inhibit transpeptidation ( Perkins , 1969 ) . Since LPS* was the product of DPP ligation onto LPS , then this modified glycoform should contain vancomycin binding sites . We assessed the ability of purified LPS* to bind vancomycin in vitro . LPS* was immobilized on a carboxymethylated dextran ( CM3 ) chip and we used surface plasmon resonance to monitor interactions with differing concentrations of vancomycin . We were able to measure specific binding of vancomycin to LPS* and to obtain a Kd = 0 . 48 ± 0 . 08 µM ( Figure 4A and Figure 4—figure supplement 1 ) , which is comparable to a reported Kd for vancomycin-lipid II interactions in vesicles ( Al-Kaddah et al . , 2010 ) . Clearly , LPS* molecules include high affinity binding sites for vancomycin . 10 . 7554/eLife . 05334 . 007Figure 4 . Mutant WaaL attaches peptidoglycan fragments to LPS . ( A ) LPS* specifically binds vancomycin . Purified LPS* was immobilized on a CM3 chip and varying concentrations of vancomycin were applied . Binding was measured at 25°C by surface plasmon resonance . Fitting of equilibrium signal yielded a Kd = 0 . 48 ± 0 . 08 µM . Standard deviation was measured for 0 . 6 µM and 1 . 2 µM and was ±1 RU . ( B ) Vancomycin binds to LPS* at the cell surface . Live exponential-phase growing cells labeled with 1 μg/ml vancomycin-BODIPY for 10 min . Cells were spotted onto M63 minimal medium agar pads and imaged by fluorescence microscopy . DOI: http://dx . doi . org/10 . 7554/eLife . 05334 . 00710 . 7554/eLife . 05334 . 008Figure 4—figure supplement 1 . SPR binding kinetics at 25°C . ( A and B ) , Different concentrations ( see legend inset in C ) of vancomycin were passed over surfaces of total isolated LPS from waaL+ ( strain MG1210 ) or total isolated LPS from waaL15 ( strain MG1211 ) . ( C ) , The reference subtracted kinetics after 400 s of injection . The equilibrium signals were plotted in Figure 4A . DOI: http://dx . doi . org/10 . 7554/eLife . 05334 . 008 The ability of LPS* to directly bind vancomycin suggested a possible resistance mechanism , namely that vancomycin is titrated outside the cell . To test this hypothesis , we performed live cell microscopy using a fluorescent vancomycin-BODIPY . We used a wild-type strain background with an intact OM that prevents the influx of vancomycin , to avoid labeling intracellular sites of PG synthesis . Indeed , waaL+ cells could not be fluorescently labeled ( Figure 4B ) . On the other hand , we readily detected circumferential labeling of waaL15 bacteria , confirming the presence of accessible D-Ala-D-Ala residues at the cell surface ( Figure 4B ) . Several vancomycin-resistance mechanisms exist in Gram-positive bacteria , including: alterations in peptidoglycan metabolism can produce thicker cell walls ( Cui et al . , 2003 ) ; and transpeptidation can be reduced to leave more free D-Ala-D-Ala residues within the established cell wall structure ( Sieradzki and Tomasz , 1997 ) . It was not immediately apparent to us that any of these strategies could be employed in E . coli since virtually all of the terminal D-Ala residues of DPP are removed either by cross-linking or by the carboxypeptidases . However , we show that the waaL15 mechanism is comparable since it also increases the number of free D-Ala-D-Ala targets that can tie up vancomycin . Moreover , by displaying D-Ala-D-Ala at the cell surface the waaL15 mutation titrates vancomycin away from the true drug target , in an altogether different cellular compartment . Therefore , LPS* confers resistance by acting as a molecular decoy for vancomycin . Given that Gram-negative bacteria are inherently resistant to vancomycin this decoy mechanism may not be of clinical significance . However , the increased resistance it does confer clearly demonstrates the tremendous adaptability of bacteria under antibiotic stress . The biosynthesis of LPS* is remarkable . Lipid II in E . coli is extremely scarce , its steady-state abundance is thought to be only 1000–2000 molecules per cell ( van Heijenoort et al . , 1992 ) . Insertion of new PG is thought to occur via large multiprotein morphogenic complexes: the elongasome and the divisome , responsible for PG synthesis along the lateral cell body and at the septum , respectively . In order to overcome the scarcity of lipid II and limit its diffusion away from sites of PG growth , both complexes are suggested to include at least some of the lipid II biosynthetic enzymes , and the presumed flippases that deliver lipid II from the site of synthesis in the cytoplasm to the site of cell wall assembly in the periplasm ( Szwedziak and Löwe , 2013 ) . In this model , the substrate for PG synthesis would be isolated physically from the LPS assembly pathway . LPS is inserted into the OM of each cell at a rate exceeding 70 , 000 molecules per minute ( Lima et al . , 2013 ) and we approximate that one-third of LPS is modified by WaaL15 with lipid II-sourced DPP . Clearly , WaaL15 has ready access to lipid II and this is inconsistent with a model of diffusion-limited lipid II sequestered at the elongasome or divisome complexes . Recent evidence also points to wider lipid II availability ( Lee et al . , 2014; Sham et al . , 2014 ) . Our data indicate that the re-charging of the lipid carrier with new DPP must also be extremely efficient to maintain such a robust pool of PG precursor . WaaL15 drains the available lipid II pool with no apparent detriment to cell wall integrity ( Figure 3—figure supplement 1 ) . Lipid II limitation can be revealed by synthetic genetic interactions in a strain lacking the elongasome ( Paradis-Bleau et al . , 2014 ) , but it is not the recharging of lipid II that is limiting , rather it is the biosynthesis of DPP ( Supplementary file 1A ) . In many bacteria , LPS is decorated with highly variable O-Ags that are linear polymers of repeating units of 3–6 monosaccharides ( Kalynych et al . , 2014 ) . In E . coli the multitude of different O-Ags initiate with GlcNAc , ECA also initiates with GlcNAc . In E . coli K-12 when colonic acid is overproduced M-LPS is made from an intitating Glc residue . The F332S mutation broadens substrate specificity of the WaaL glycosyltransferase allowing it to efficiently accept a significantly more bulky initiating MurNAc with an attached oligopeptide stem . The only other glycosyltransferase that is known to use lipid II as a substrate is PglL from Neisseria and the use required overproduction of the enzyme in E . coli ( Faridmoayer et al . , 2008 ) . It is also remarkable that we detect no OM biogenesis defect in strains carrying waaL15 , demonstrating that the Lpt system is fully competent for the transport and assembly of LPS* despite the addition of both unnatural sugars and peptide stems . Both LPS and PG are pathogen-associated molecular patterns ( PAMPs ) that potently activate innate immune responses via distinct pathways , and it seems sensible for Gram-negative bacteria to keep these entities separated . We suggest that the F332S substitution has inactivated an exclusion mechanism that prevents WaaL from utilizing the lipid II pool .
Strains and plasmids used in this study are listed in Supplementary file 1B and Supplementary file 1C , respectively . Strains were grown in lysogeny broth ( LB , 10 g/L NaCl ) or M63 minimal medium under aeration at 37°C unless otherwise noted . When appropriate , media were supplemented with kanamycin ( Kan , 25 μg/ml ) , ampicillin ( Amp , 25–125 μg/ml ) , tetracycline ( Tet , 20 μg/ml ) , chloramphenicol ( Cam , 20 μg/ml ) , vancomycin ( Vanc , 65–260 μg/ml ) and arabinose ( Ara , 0 . 2% vol/vol ) . Kanamycin deletion-insertion mutations of bamE , cpsG , mrcA , mrcB , lpoA and lpoB were obtained from the Keio collection ( Baba et al . , 2006 ) . ECA null rff::Tn10-66 allele was obtained from strain 21566 ( Meier-Dieter et al . , 1990 ) . The ompC::Tn5 rcsC137 was obtained from strain SG20803 ( Brill et al . , 1988 ) . Mutant alleles were introduced by P1vir transduction . Spontaneous suppressor mutants of strain MG1029 capable of growing on LB plates supplemented with vancomycin ( 140 μg/ml ) were isolated; one such mutant strain was MG1088 . The mutation locus conferring vancomycin-resistance in MG1088 was identified by linkage mapping using a library of random mini-Tn10 insertions ( Kleckner et al . , 1991 ) . In this way , the tdh::Tn10 allele was found to be approximately 70% linked to the suppressor mutation waaL15 . The F332S mutation was then identified by PCR amplification and sequencing of the waaL locus . The waaL15 mutation was moved into the NR754 wild-type strain by linkage with tdh::Tn10 . In order to generate the unmarked waaL15 strain ( MG1643 ) and its wild-type control ( MG1642 ) , the tdh::Tn10 mutation was removed from strains MG1210 and MG1211 by first introducing a linked ΔcysE::kan mutation ( Baba et al . , 2006 ) , selecting for KanR and screening for TetS transductants that were VancR ( waaL15 ) or VancS ( waaL+ ) . The ΔcysE::kan mutation was then replaced with cysE+ by transduction , selection on M63 minimal medium , and screening of VancR/VancS . In E . coli , two key PG synthases , PBP1A ( mrcA/ponA ) and PBP1B ( mrcB/ponB ) , incorporate DPP from Lipid II into PG strands and also mediate transpeptidation ( Paradis-Bleau et al . , 2010; Typas et al . , 2010 ) . Recent evidence suggests that lipid II limitation can be revealed by synthetic genetic interactions in a strain lacking mrcB ( Paradis-Bleau et al . , 2014 ) . The genetic interaction of PG synthase mutants with waaL15 was assessed as follows . KanR-marked null alleles of lpoA , lpoB , mrcA and mrcB were introduced by P1vir transduction into CAG strains that carry a Tn10 insertion in a nearby locus ( see Supplementary file 1A ) . KanR TetR transductants were isolated and used to generate P1vir lysates . These P1vir were used to transduce waaL+ ( MG1642 ) or waaL15 ( MG1643 ) strains , selecting for the Tn10 marker . The frequency with which the KanR-marked lpo and mrc alleles were co-transduced ( genetically linked ) was determined by replica plating on LB+Kan . Linkage was assessed in a total of 300 transductants from three independent experiments . A decrease in the cotransduction frequency in waaL15 strains relative to waaL+ indicates a synthetic interaction between waaL15 and the KanR-marked allele . The synthetic interaction between waaL15 and mrcB/lpoB null alleles was relieved in strains carrying pMurA when expression of the cloned murA gene ( encoding the enzyme responsible for the first committed step in DPP biosynthesis ) was induced with 100 μM isopropyl β-D-1-thiogalactopyranoside ( IPTG ) . Overexpression of murA increases the cellular pool of UDP-MurNAc-pentapeptide and consequently also increases the pool of lipid II . A total to 1 × 109 cells from liquid culture were taken , pelleted and resuspended 0 . 05 ml of ‘LPS Sample Buffer’ ( 0 . 66 M Tris pH 7 . 6 , 2% vol/vol sodium dodecyl sulfate [SDS] , 10% vol/vol glycerol , 4% vol/vol β-mercaptoethanol , 0 . 1% wt/vol bromophenol blue ) . Samples were boiled for 10 min and allowed to cool to room temperature . 10 μl of Proteinase K ( 2 . 5 mg/ml , in LPS Sample Buffer ) was added and samples were incubated at 56°C for 16 hr . LPS samples were then resolved by SDS-PAGE and silver stained as described previously ( Tsai and Frasch , 1981 ) . By quantifying band density using ImageJ , we measured that LPS* constitutes 29 ± 1% of the total LPS in waaL15 samples . 3 ml of molten LB Top agar ( 0 . 75% agar ) was inoculated with 0 . 1 ml of overnight culture . The mixture was poured onto a LB agar plate ( 1 . 5% agar , ) and allowed to set . Antibiotic discs ( BD Sensi-Disc ) were placed on the Top agar overlay and plates were incubated overnight at 37°C . The ‘zone of growth inhibition’ was measured across the antibiotic disc . Overnight cultures were sub-cultured at 1:100 into fresh LB broth and grown for 1 . 5 hr . A 1 ml aliquot was taken , pelleted and was twice washed with 1 ml M63 medium . Cells were resuspended in 0 . 1 ml of M63 broth containing 1 μg/ml of vancomycin-BODIPY-FL ( Life Technologies , V-34850 ) . Cells were incubated at room temperature for 10 min and then washed twice with 1 ml M63 broth . Cells were then resuspended in 0 . 03 ml of M63 broth , and approximately 2 ml was spotted onto an M63-agarose pad . Cells were immediately visualized on a Nikon Eclipse 90i microscope with a Nikon Plan Apo 1 . 4/100 × Oil Ph3 phase objective . E . coli MG1210 and MG1211 were each grown in 4 × 1 . 5 l LB medium shaking at 37°C overnight to stationary phase . The cells were harvested by centrifugation for 15 min at 5000×g , 4°C and washed with water ( 700 ml ) and ethanol ( 40 ml ) once , then twice with acetone ( 40 ml ) . After drying the cell pellet in a desiccator over night in vacuo , PCP ( Phenol-Chloroform-petroleum ether ) method was used for rough LPS extraction ( Galanos et al . , 1969 ) . E . coli MG1210 and MG1211 were each grown in 500 ml LB medium shaking at 37°C to stationary phase ( 6 hr ) . The cell wall was isolated from the culture as described by Glauner et al . ( 1988 ) and Uehara et al . ( 2009 ) , with modifications described below . The cells were resuspended in 20 ml phosphate buffered saline ( PB , pH = 7 . 4 ) and boiled for 30 min in 80 ml 5% SDS . After the samples cooled , they were pelleted ( 14 , 000 rpm , 25°C , 1 hr ) and washed six times by pelleting ( 14 , 000 rpm , 25°C , 1 hr ) from 50 ml water aliquots to remove the SDS . The samples were resuspended in 1 ml PBS , treated with α-amidase ( 100 µl , 2 mg/ml stock in 50% glycerol , Sigma A-6380 ) and incubated at 37°C with shaking for 2 hr . To cleave proteins attached to the cell wall , α-chymotrypsin ( 100 µl , 3 mg/ml in 50% glycerol , Sigma C3142 ) was added , and the samples were incubated at 37°C with shaking overnight . An additional aliquot of α-chymotrypsin ( 100 µl ) was added , and the samples were digested for an additional 4 hr . To remove the proteins , SDS was added to a final concentration of 1% , and the samples were incubated at 95°C for 1 hr . After cooling , the samples were again pelleted ( 14 , 000 rpm , 25°C , 1 hr ) and washed with water repeatedly ( 4 × 25 ml ) to remove the SDS . The final peptidoglycan ( PG ) samples were resuspended in 500 µl 0 . 02% azide and stored at 4°C . The PG composition was analyzed by LC/MS as previously described ( Lebar et al . , 2013 ) . The method was also used to analyze LPS samples . The glycosylhydrolase mutanolysin liberated DPP and disaccharide tetrapeptide from LPS* . Aliquots ( 40 µl ) of PG ( from MG1210 and MG1211 ) and LPS ( from MG1210 and MG1211 ) were incubated with mutanolysin ( 10 U , 2 . 5 µl , 4000 U/ml , Sigma M9901 , stored at −20°C in 50 mM TES , pH 7 . 0 , 1 mM MgCl2 , 10% glycerol ) in 50 mM sodium phosphate buffer ( pH 6 . 0 , 100 μl total volume ) at 37°C with shaking overnight . Another aliquot of mutanolysin ( 10 U , 2 . 5 µl ) was added , and the mixture was incubated at 37°C with shaking for 3 hr . Insoluble particles were separated by centrifugation ( 16 , 000×g ) . The supernatant , containing soluble fragments , was treated with sodium borohydride ( 10 mg/ml in water , 100 μl ) at room temperature for 30 min . Phosphoric acid ( 20% , 12 μl ) was then added to adjust pH to ∼4 . When bubbling ceased , the samples were lyophilized and re-dissolved in 25 μl water , which was analyzed on LC/MS . LC/MS analysis was conducted with ESI-MS operating in positive mode . The instrument was equipped with a Waters Symmetry Shield RP18 column ( 5 μm , 3 . 9 × 150 mm ) with matching column guard . The fragments were separated using the following method: 0 . 5 ml/min H2O ( 0 . 1% formic acid ) for 5 min followed by a gradient of 0% ACN ( 0 . 1% formic acid ) /H2O ( 0 . 1% formic acid ) to 20% ACN ( 0 . 1% formic acid ) /H2O ( 0 . 1% formic acid ) over 40 min . Purified LPS ( 0 . 5 mg/ml ) from strains MG1210 or MG1211 were extruded in 20 mM Tris/HCl pH 8 , 150 mM NaCl and immobilized on poly-L-lysine coated CM3 Biacore chips on the active and reference channel , respectively ( Malojčić et al . , 2014 ) . All experiments were performed using a Biacore X100 instrument at 25°C at a flow rate of 10 μl/min with 20 mM Tris/HCl pH 8 , 150 mM NaCl buffer . Different concentrations of vancomycin were injected for 400 s and dissociation was recorded for another 500 s to return to baseline . No binding was observed to the reference channel . The equilibrium signal in the difference channel was fitted to f = Bmax*abs ( x ) / ( Kd + abs ( x ) ) with R2 = 0 . 88 . Standard deviation was measured for 0 . 6 µM and 1 . 2 µM vancomycin in triplicate and did not exceed 1 RU .
The lipopolysaccharide ( LPS ) forms the surface-exposed leaflet of the outer membrane ( OM ) of Gram-negative bacteria , an organelle that shields the underlying peptidoglycan ( PG ) cell wall . Both LPS and PG are essential cell envelope components that are synthesized independently and assembled by dedicated transenvelope multiprotein complexes . We have identified a point-mutation in the gene for O-antigen ligase ( WaaL ) in Escherichia coli that causes LPS to be modified with PG subunits , intersecting these two pathways . Synthesis of the PG-modified LPS ( LPS* ) requires ready access to the small PG precursor pool but does not weaken cell wall integrity , challenging models of precursor sequestration at PG assembly machinery . LPS* is efficiently transported to the cell surface without impairing OM function . Because LPS* contains the canonical vancomycin binding site , these surface-exposed molecules confer increased vancomycin-resistance by functioning as molecular decoys that titrate the antibiotic away from its intracellular target . This unexpected LPS glycosylation fuses two potent pathogen-associated molecular patterns ( PAMPs ) .
Tiny Gram-negative bacteria are one of humankind's deadliest foes , causing infections of wounds and the bloodstream that are very hard to treat . Many Gram-negative bacteria are resistant to several common antibiotics , and the few treatments available that can successfully kill the bacteria are often also toxic to the patients . Understanding how these bacteria elude antibiotics could help scientists develop better , less toxic treatments . Most bacteria are surrounded by a cell wall that helps protect the bacteria and gives them structure . Many broad-spectrum antibiotics , including penicillin and vancomycin , work by interfering with how this protective wall is built from molecules called peptidoglycans . However , Gram-negative bacteria have an outer membrane that prevents many antibiotics from reaching the cell wall , and so the antibiotics are unable to kill the bacteria . The outer membrane of Gram-negative bacteria is made up of sugars and fatty molecules called lipids . Recently , scientists discovered a mutation that interferes with the movement of the lipid and sugar molecules that make up the outer membrane , which compromises this protective layer and makes the bacteria more susceptible to antibiotics . To learn more about how this mutation interferes with the defenses of the Gram-negative bacteria Escherichia coli , Grabowicz et al . searched for compensating mutations that can counteract it and restore the antibiotic resistance of these mutant bacteria . The search revealed that a mutation in a gene called waaL increases E . coli's resistance to vancomycin , but not to other antibiotics . The gene encodes an enzyme , and the mutant form of the enzyme attaches some peptidoglycans to the surface of the outer membrane instead of incorporating them into the cell wall . The stray peptidoglycans on the cell's surface act as decoys , binding to vancomycin and keeping the drug from reaching its true target—the cell wall . The decoy strategy is similar to a mechanism used by Gram-positive bacteria—which lack a protective outer membrane—to resist vancomycin treatment , which also involves creating sites that bind the drug and keep it from its target . Vancomycin is not currently used clinically to treat E . coli or other Gram-negative infections because these bacteria are naturally quite resistant for other reasons . However , Grabowicz et al . 's findings do demonstrate how quickly bacteria can adapt and produce new defenses to antibiotics when old strategies fail .