Identification of MupP as a NewPeptidoglycan Recycling Factor andAntibiotic Resistance Determinant inPseudomonas aeruginosa

Coralie Fumeaux, Thomas G. BernhardtDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, USA

ABSTRACT Peptidoglycan (PG) is an essential cross-linked polymer that surroundsmost bacterial cells to prevent osmotic rupture of the cytoplasmic membrane. Itssynthesis relies on penicillin-binding proteins, the targets of beta-lactam antibiotics.Many Gram-negative bacteria, including the opportunistic pathogen Pseudomonasaeruginosa, are resistant to beta-lactams because of a chromosomally encoded beta-lactamase called AmpC. In P. aeruginosa, expression of the ampC gene is tightly reg-ulated and its induction is linked to cell wall stress. We reasoned that a reportergene fusion to the ampC promoter would allow us to identify mutants defective inmaintaining cell wall homeostasis and thereby uncover new factors involved in theprocess. A library of transposon-mutagenized P. aeruginosa was therefore screenedfor mutants with elevated ampC promoter activity. As an indication that the screenwas working as expected, mutants with transposons disrupting the dacB gene wereisolated. Defects in DacB have previously been implicated in ampC induction andclinical resistance to beta-lactam antibiotics. The screen also uncovered murU andPA3172 mutants that, upon further characterization, displayed nearly identical drugresistance and sensitivity profiles. We present genetic evidence that PA3172, re-named mupP, encodes the missing phosphatase predicted to function in the MurUPG recycling pathway that is widely distributed among Gram-negative bacteria.

IMPORTANCE The cell wall biogenesis pathway is the target of many of our bestantibiotics, including penicillin and related beta-lactam drugs. Resistance to thesetherapies is on the rise, particularly among Gram-negative species like Pseudomonasaeruginosa, a problematic opportunistic pathogen. To better understand how theseorganisms resist cell wall-targeting antibiotics, we screened for P. aeruginosa mu-tants defective in maintaining cell wall homeostasis. The screen identified a new fac-tor, called MupP, involved in the recycling of cell wall turnover products. Character-ization of MupP and other components of the pathway revealed that cell wallrecycling plays important roles in both the resistance and the sensitivity of P. aerugi-nosa to cell wall-targeting antibiotics.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen capable ofgrowth in diverse environments (1). In hospitals, it causes a number of serious

infections (2, 3). The key drugs in our arsenal for treating these infections are thebeta-lactam antibiotics, including cephalosporins, monobactams, and carbapenems,which target the biogenesis of the peptidoglycan (PG) cell wall (4). Resistance to theseantibiotics is on the rise among Gram-negative bacteria like P. aeruginosa and is oftenassociated with multidrug resistance phenotypes. A frequent mechanism of resistanceto beta-lactams is overproduction of the chromosomally encoded beta-lactamasecalled AmpC, which inactivates penicillins, cephalosporins, and monobactams (5–8).

Received 23 January 2017 Accepted 6 March2017 Published 28 March 2017

Citation Fumeaux C, Bernhardt TG. 2017.Identification of MupP as a new peptidoglycanrecycling factor and antibiotic resistancedeterminant in Pseudomonas aeruginosa. mBio8:e00102-17. https://doi.org/10.1128/mBio.00102-17.

Editor Howard A. Shuman, University ofChicago

Copyright © 2017 Fumeaux and Bernhardt.This is an open-access article distributed underthe terms of the Creative Commons Attribution4.0 International license.

Address correspondence to Thomas G.Bernhardt, thomas_bernhardt@hms.harvard.edu.

For a companion article on this topic, seehttps://doi.org/10.1128/mBio.00092-17.

RESEARCH ARTICLE

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AmpC is a broadly distributed group I, class C cephalosporinase produced by mostEnterobacteriaceae family members and many nonfermenting Gram-negative bacilli inaddition to P. aeruginosa (9). In the absence of stress, AmpC production is relatively lowin wild-type strains (10). However, in the presence of certain beta-lactams, such ascefoxitin (Fox) and imipenem (beta-lactamase inducers), ampC expression is highlyactivated (10). Although they are sensitive to hydrolysis by AmpC, antipseudomonalpenicillins like piperacillin (Pip) and cephalosporins like ceftazidime (Caz) are effectivebecause they avoid ampC induction (11). However, mutants defective in ampC regu-lation that constitutively produce high levels of beta-lactamase have been isolated inthe clinic and can cause failures of antimicrobial therapy (7, 12–16).

The mechanism of ampC regulation is intimately connected to the PG synthesis and re-cycling pathways (Fig. 1) (17). PG synthesis begins in the cytoplasm with the formationof UDP–N-acetylmuramic acid (UDP-MurNAc) from UDP–N-acetylglucosamine (UDP-GlcNAc) through the action of the enzymes MurA and MurB. A pentapeptide (pep5) isadded to UDP-MurNAc in several steps, forming UDP-MurNAc-pep5. The phospho-MurNAc-pep5 moiety of this intermediate is then transferred to the lipid carrierundecaprenol phosphate (Und-P), forming lipid I. GlcNAc from UDP-GlcNAc is thenadded to form lipid II, which is the final precursor and contains the MurNAc-pep5-GlcNAc monomeric unit of PG. After lipid II is translocated (18) to expose thedisaccharide-peptide on the outer surface of the cytoplasmic membrane, it is polym-erized and cross-linked into the PG layer by penicillin-binding proteins (PBPs) (19) andSEDS family proteins (20) to expand the existing matrix.

Far from being inert, the PG layer is constantly remodeled during cell growth.Roughly 40% of the PG layer is turned over per generation in Escherichia coli (21). Theliberated fragments are primarily generated by the action of endopeptidases (EPs) thatcleave the peptide cross-links and lytic transglycosylases (LTs) that cleave the sugarbackbone. Rather than hydrolyzing the glycans, LTs promote the formation of 1,6-anhydro linkages in MurNAc such that the main PG degradation products released fromthe matrix are GlcNAc-1,6-anhMurNAc peptides (21) (Fig. 1). These anhydro-muro-peptides are subsequently transported into the cytoplasm by the permease AmpG (22)and possibly AmpP in P. aeruginosa (23), where they are further broken down into theirbasic components by a succession of enzymes (21, 24) (Fig. 1). The glycosidase NagZremoves the GlcNAc moiety (25, 26), and the amidase AmpD removes the stem peptidefrom the NagZ-processed product or the incoming disaccharide (27, 28). The releasedpeptides are further processed to tripeptides by the L,D-carboxypeptidase LdcA andreattached to UDP-MurNAc for recycling by Mpl (29, 30) (Fig. 1). Recycling of the PGsugars is carried out by one of two possible pathways in Gram-negative bacteria (Fig. 1).The first pathway was discovered in E. coli and ultimately converts GlcNAc and1,6-anhMurNAc to glucosamine-1-phosphate (GlcN-1P) for the regeneration of UDP-GlcNAc by the de novo biosynthesis pathway involving GlmU (21, 31, 32) (Fig. 1). Thesecond pathway was discovered recently and is more broadly conserved amongGram-negative bacteria, including P. aeruginosa (33). It uses the enzymes AmgK andMurU to more directly convert 1,6-anhMurNAc back to UDP-MurNAc, thus bypassing denovo biosynthesis (33, 34).

The main regulator of ampC expression is AmpR. In nonstressed cells, it associateswith the PG precursor UDP-MurNAc-pep5 and functions as a repressor (35, 36). Beta-lactams inhibit PG cross-linking by the PBPs, causing the formation of uncross-linkedglycans that are rapidly degraded by LTs into turnover products (37). The resultingaccumulation of anhydro-muropeptides in the cytoplasm is thought to compete withUDP-MurNAc-pep5 for binding to AmpR and convert the regulator into an activator ofampC transcription (10, 38–41). Following AmpC production and export to theperiplasm, the beta-lactam molecules are inactivated by hydrolysis and homeostasis isrestored, eventually resulting in a decrease in cytoplasmic anhydro-muropeptide levelsand repression of ampC (42).

Because it functions as a key sensor of PG homeostasis, we reasoned that an ampCpromoter fusion to lacZ might serve as a useful tool to identify new P. aeruginosa

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factors involved in cell wall synthesis, repair, and recycling. To this end, we mu-tagenized a strain encoding a chromosomally integrated PampC::lacZ fusion (43) with atransposon and plated the resulting mutant library on plates containing X-Gal (5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside). Colonies displaying increased bluecolor, indicative of PampC::lacZ induction, were isolated, and the locations of transposoninsertions in these isolates were mapped. As an indication that the screen was workingas expected, mutants with transposons disrupting dacB were isolated. DacB defectshave previously been implicated in ampC induction and clinical resistance to beta-lactam antibiotics (7, 14). The screen also uncovered murU and PA3172 mutants that,upon further characterization, displayed nearly identical drug resistance and sensitivityprofiles. We present genetic evidence that PA3172, renamed mupP, encodes the missing

flippase PP GP MP M

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FIG 1 Simplified pathways for PG synthesis and recycling and the link to ampC regulation. (A) The PG matrixconsists of glycan chains with the repeating unit of MurNAc (M) and GlcNAc (G). Attached to the MurNAc sugarsis a pep5 (L-Ala-�-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala, colored circles) used to form cross-links betweenadjacent glycans. PG synthesis starts in the cytoplasm, is continued by the generation of lipid-linked precursors,and ends with the polymerization and cross-linking reactions at the membrane surface to build PG. The matrix isalso subject to degradation by LTs and EPs to generate anhMurNAc-containing turnover products, which arerecycled. The names of the general recycling enzymes present in both E. coli and P. aeruginosa are black. Theproteins found uniquely in E. coli and in P. aeruginosa are red and blue, respectively. See the text for details. (B)Under normal conditions (no drug, left side), the PG precursor UDP-MurNAc-pep5 binds to AmpR and causesrepression of ampC transcription (35, 36). During beta-lactam stress (right side), PG cross-linking is blocked andturnover is elevated (37). This imbalance causes accumulation of anhMurNAc-pep5 and GlcNAc-anhMurNAc-pep5in the cytoplasm. The accumulated anhydro-muropeptides are thought to competitively displace UDP-MurNAc-pep5 from AmpR and convert it into an activator of ampC transcription (10, 38, 40, 41).

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phosphatase enzyme previously predicted (33) to function in the broadly distributedMurU pathway for PG recycling. Biochemical results in a parallel study by the Mayergroup support this designation (44).

RESULTSIdentification of transposon mutants that induce ampC expression. To identify

new factors involved in PG homeostasis, recycling, and remodeling, we took advantageof the connection between ampC induction and cell wall stress (10, 28). A strain bearinga PampC::lacZ expression construct at the attB locus (43) was generated to search formutants displaying a constitutive ampC induction phenotype. To test the activity of thereporter and its responsiveness to cell wall defects, we deleted the dacB gene in thereporter strain. DacB is a cell wall carboxypeptidase that trims the peptide within PG(45, 46). Its inactivation was previously shown to cause constitutive expression of ampC(7). As expected, the ΔdacB mutant reporter strain formed dark blue colonies on LB agarcontaining X-Gal. Reporter activity in this background was abolished upon inactivationof the AmpG permease, indicating that PampC::lacZ induction in the ΔdacB backgroundrequires the import of PG turnover products, as has been shown previously for thenative ampC locus (47). On the basis of its behavior in these mutant backgrounds, weconcluded that the PampC::lacZ reporter strain was functional and appropriate for use inscreening for cell wall homeostasis mutants.

Cells of the reporter strain CF263 (PAO1 PampC::lacZ) were mutagenized with a trans-poson carrying a tetracycline (Tet) resistance cassette that was delivered by conjugationfrom E. coli. The resulting mutant library was then plated on agar containing X-Gal toidentify constitutive PampC mutants. Colonies displaying increased blue color, indicativeof lacZ induction, arose at a frequency of approximately 10�5. Following purification,isolates were grown in liquid medium to measure beta-galactosidase activity relative tothat of the parental strain. The transposon insertion sites were then mapped for strainsconfirmed to have elevated lacZ expression. As an indication that the screen wasworking as expected, two mutants were isolated that each possessed a differentinsertion in the dacB gene. In addition to these strongly induced alleles, we also isolatedmutants that formed light blue colonies on X-Gal agar and had mildly elevatedbeta-galactosidase activity (Fig. 2). Mapping revealed that these isolates had trans-poson insertions in the murU and PA3172 genes. The absence of ampD mutants (14)among our isolates indicates that the screen is not yet saturated and further screeningshould yield additional mutants that activate the ampC reporter.

MurU is an �-1-phosphate uridyl transferase that converts MurNAc-1P to UDP-MurNAc in the Pseudomonas PG recycling pathway (33) (Fig. 1). The PA3172 gene isannotated as encoding a phosphoglycolate phosphatase, and its product was found topossess phosphatase activity against small-molecule substrates with a phosphatemoiety (48). This activity of PA3172 was intriguing because a phosphatase was previ-ously predicted to function in the MurU PG recycling pathway but has remainedunidentified (33) (Fig. 1). Because of its biochemical activity and the similar PampC::lacZinduction phenotypes displayed by mutants with murU and PA3172 inactivated, wehypothesized that PA3172 may encode the missing recycling phosphatase. Resultspresented below and those from a parallel study by the Mayer group (44) support thishypothesis. We therefore have renamed the PA3172 gene mupP for MurNAc-6P phos-phatase.

Deletion of mupP increases ampC expression and promotes beta-lactam resis-tance similar to other PG recycling mutants. To confirm their involvement in ampCoverexpression, in-frame deletions of murU and mupP were generated in the reporterstrain along with deletions in genes coding for other members of the MurU recyclingpathway (anmK and amgK). When these mutants were spotted onto agar containingX-Gal, they gave rise to zones of growth with a light blue color relative to wild-type orΔdacB mutant cells, which appeared white or dark blue, respectively (Fig. 2A). Quan-tification of beta-galactosidase activity confirmed that mutants defective for mupPdisplayed a similar level of lacZ expression as a ΔmurU mutant strain (Fig. 2B). To

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monitor the effects of these mutations on native ampC induction, the set of deletionsin mupP and recycling genes was also generated in an otherwise wild-type background.The deletion strains all showed elevated resistance to the antipseudomonal beta-lactams Caz and cefotaxime (Ctx), with resistance being intermediate compared to thatof a ΔdacB mutant (Fig. 3A). Normal beta-lactam sensitivity was restored to ΔmurU andΔmupP mutant cells by the expression of the corresponding gene from a plasmid(Fig. 3B), indicating that the phenotype was caused by the inactivation of MurU orMupP and was not an effect of the deletions on the expression of nearby genes.Elevated drug resistance in ΔmurU and ΔmupP mutant cells was dependent on ampCand its transcriptional regulator ampR (Fig. 4), consistent with resistance arising fromampC induction. Finally, ampC induction in the recycling mutants was confirmed bydirectly measuring basal levels of AmpC enzymatic activity by using the reportersubstrate nitrocefin (Fig. 5). Notably, inactivation of MupP yielded a level of AmpCactivity in cell extracts equivalent to that of strains with defects in the known recyclingenzymes MurU, AnmK, and AmgK (Fig. 5A). These strains also retained the ability toinduce high levels of AmpC production in response to treatment with the stronginducer Fox (Fig. 5B). As expected from the intermediate drug resistance phenotype,the level of induction of the recycling-defective strains was much less than that of thehighly resistant ΔdacB mutant. We conclude that mutants with the MurU recyclingpathway disrupted have elevated beta-lactam resistance because of ampC inductionand that mutants with defects in MupP share this phenotype.

MupP-defective strains are Fos hypersensitive. Strains with the recycling genemurU, amgK or anmK inactivated were previously shown to be hypersensitive to theantibiotic fosfomycin (Fos) (33, 34). This drug targets MurA activity and thus blocks theconversion of UDP-GlcNAc into UDP-MurNAc as part of the de novo PG precursorsynthesis pathway (Fig. 1) (49). A functional MurU pathway bypasses MurA in theconversion of cell wall turnover products into UDP-MurNAc (Fig. 1). It therefore reducesthe need for MurA activity, thereby increasing Fos resistance. We reasoned that if MupPis indeed part of the MurU pathway, its inactivation should also result in Fos hyper-sensitivity. Plating of serial dilutions of ΔmupP mutant cells on LB agar with or without

FIG 2 PampC::lacZ expression in mupP and murU deletion strains. (A) Cultures (5 �l) of strains PAO1 (wildtype [WT]), CF268 (ΔdacB mutant), CF706 (ΔanmK mutant), CF594 (ΔmupP mutant), CF600 (ΔamgKmutant), and CF485 (ΔmurU mutant) containing the PampC::lacZ reporter were spotted onto LB agarcontaining X-Gal (50 �g/ml), grown overnight at 30°C, and photographed. (B) �-Galactosidase activitywas measured in liquid cultures of the strains indicated. The activity in the wild-type strain was set at100%, and the activity in the other strains is reported relative to wild-type activity. Results shown are theaverages of three assays with two biological replicates per strain, and the error bars represent thestandard deviation. *, P � 0.01; **, P � 0.0001 (compared to wild-type expression, as determined byWelch’s unequal-variance t test).

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Fos revealed a hypersensitivity phenotype that mimicked that of mutants with othercomponents of the MurU pathway deleted (Fig. 6A). As with a murU mutant, normal Fosresistance was restored to the ΔmupP mutant strain by expression of the mupP gene intrans from a plasmid (Fig. 6B). This result reinforces the phenotypic similarity of ΔmupP

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FIG 3 Beta-lactam resistance of strains with PG recycling factors deleted. (A) Cultures of strains PAO1(wild type [WT]), CF155 (ΔdacB mutant), CF550 (ΔanmK mutant), CF592 (ΔmupP mutant), CF596 (ΔamgKmutant), and CF488 (ΔmurU mutant) were serially diluted, and 5 �l of each dilution was spotted onto LBagar supplemented with Caz (4 �g/ml) or Ctx (25 �g/ml), as indicated. The Caz and Ctx MICs determinedby agar dilution were 2.5 and 25 �g/ml for the wild type and 5 and 30 �g/ml for the recycling mutants,respectively. An increase in the MICs for the recycling mutants was not observed in liquid medium. (B)Cultures of CF732 (PAO1 [empty]), CF155 (ΔdacB mutant), CF521 (ΔmupP [empty]), CF505 (ΔmupP[Plac::mupP]), CF517 (ΔmurU [empty]), and CF519 (ΔmurU [Plac::murU]) were serially diluted and plated onLB agar supplemented with IPTG (1 mM), Caz (4 �g/ml), or both, as indicated. Expression constructs wereintegrated at the attTn7 locus.

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FIG 4 AmpR and AmpC are required for the beta-lactam resistance phenotype of ΔmurU and ΔmupPmutant strains. Cultures of strains PAO1 (wild type [WT]), CF155 (ΔdacB) mutant, CF488 (ΔmurU mutant),CF690 (ΔmurU ΔampC mutant), CF608 (ΔmurU ΔampR mutant), CF592 (ΔmupP mutant), CF692(ΔmupPΔampC mutant), and CF647 (ΔmupPΔampR mutant) were serially diluted, and 5 �l of eachdilution was spotted onto LB agar with or without Ctx (25 �g/ml), as indicated.

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mutant cells and mutants with changes in known components of the MurU recyclingpathway.

Expression of mupP allows reconstitution of the full MurU pathway in E. coli.E. coli lacks the MurU pathway and is therefore relatively sensitive to Fos. Instead, it usesthe MurQ enzyme to convert MurNAc-6P to GlcNAc-6P for reentry into the de novopathway (Fig. 1). In a ΔmurQ mutant, MurNAc recycling is blocked at MurNAc-6P. TheMayer group was previously able to partially reconstitute the P. putida MurU pathwayin an E. coli ΔmurQ mutant, as assessed by increased Fos resistance (33). They did so byexpressing amgK and murU from a plasmid. Because the MurNAc-6P phosphataseremained unidentified at the time, Fos resistance was only restored by supplyingMurNAc in the medium for uptake and entry into the pathway. This result suggestedthat the E. coli ΔmurQ mutant cells were unable to process endogenous MurNAc-6P foruse in the recycling pathway by amgK and murU. Thus, if MupP is indeed theMurNAc-6P phosphatase in the MurU pathway, coexpression of mupP with amgK andmurU in E. coli ΔmurQ mutant cells should result in increased Fos resistance without theneed for externally added MurNAc. Indeed, expression of wild-type mupP in conjunc-tion with amgK and murU promoted increased Fos resistance to E. coli ΔmurQ mutantcells. Increased resistance was not observed when mupP was expressed alone or whena predicted MupP catalytic mutant protein, MupP(D12A) (48), was produced in tandemwith AmgK and MurU (Fig. 7). On the basis of these results and the similar phenotypes

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FIG 5 AmpC activity in strains with PG recycling factors deleted. Assay of nitrocefin hydrolysis by cellsof PAO1 (wild type [WT]), CF155 (ΔdacB mutant), CF550 (ΔanmK mutant), CF592 (ΔmupP mutant), CF596(ΔamgK mutant), and CF488 (ΔmurU mutant) grown in LB (A) or LB supplemented with 50 �g/ml Fox (B).The ΔdacB mutant served as the positive control and has highly elevated basal AmpC activity, while therecycling mutants have slightly increased activity compared to that of the wild type (PAO1). BSA and theno-protein control have no detectable AmpC activity. Data are the mean of three independent assayseach for two biological replicates with the error bars indicating the standard error. *, P value � 0.01compared to wild-type AmpC activity, as determined by Welch’s unequal-variance t test.

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displayed by mupP mutants and mutants defective in PG recycling, we conclude thatMupP is the missing phosphatase acting in the MurU pathway. Consistent with thisconclusion, MupP is co-conserved with AmgK and MurU in a range of proteobacteriabut absent in others like the enterobacteria that lack the MurU pathway (Fig. 8).

DISCUSSION

Many Gram-negative bacteria encode an inducible AmpC beta-lactamase that pro-vides resistance to beta-lactam antibiotics (42). The ampC gene is normally repressed byAmpR when cell wall biogenesis is proceeding normally but is expressed when anelevated level of PG turnover products accumulates in the cytoplasm as a result of abeta-lactam-induced block in PG cross-linking (35–37). Thus, expression of ampC istuned to respond when the balance of cell wall synthesis and degradation is upset. Wetherefore employed a lacZ reporter fused to the ampC promoter in P. aeruginosa to

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FIG 7 Reconstitution of the complete MurU pathway in E. coli. E. coli strain CF752 (MG1655 ΔmurQ) harboring pUC18 (vector)or pCF436 (Plac::amgK-murU) along with the compatible vector pCF826 (Plac::mupP) or pCF836 (Plac::mupP[D12A]), as indicated,was serially diluted, and 5 �l of each dilution was spotted onto LB agar supplemented with Fos (2 �g/ml), IPTG (100 �M), orboth, as indicated. WT, wild type.

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FIG 6 Fos sensitivity of a ΔmupP mutant. (A) Cultures of strains PAO1 (wild type [WT]), CF155 (ΔdacBmutant), CF550 (ΔanmK mutant), CF592 (ΔmupP mutant), CF596 (ΔamgK mutant), and CF488 (ΔmurUmutant) were serially diluted, and 5 �l of each dilution was spotted onto LB agar with or without Fos(25 �g/ml), as indicated. The Fos MIC was determined by broth dilution and is �40 �g/ml for the wildtype and 15 �g/ml for the recycling mutants, respectively. (B) Cultures of CF732 (PAO1[empty]), CF521(ΔmupP [empty]), CF505 (ΔmupP [Plac::mupP]), CF517 (ΔmurU [empty]), and CF519 (ΔmurU [Plac::murU])were serially diluted on LB agar as described for panel A. LB agar was supplemented with 1 mM IPTG,Fos (25 �g/ml), or both, as indicated. Expression constructs were integrated at the attTn7 locus.

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nloaded from

screen for mutants with PG homeostasis defects with the goal of identifying newfactors involved in the process. The screen was successful and identified mupP (PA3172),a gene of previously unknown function, as encoding a new enzyme involved in PGrecycling.

Recycling of PG turnover products in Gram-negative bacteria is carried out by oneof two possible pathways, (i) the MurQ pathway used by E. coli and its relatives, in whichthe sugars of PG turnover products are funneled back into the de novo PG precursorsynthesis pathway, or (ii) the MurU pathway, which more directly converts MurNAcfrom PG turnover products to UDP-MurNAc and bypasses de novo synthesis (Fig. 1) (33).Transposon insertion or mupP deletion mutant strains displayed ampC inductionphenotypes that were identical to those of mutants defective for MurU and othermembers of the MurU pathway. Additionally, co-expression of mupP with murU andamgK was sufficient to reconstitute the MurU pathway in E. coli, which is normally

Variovorax sp DB1

Mar

inom

onas

pos

idon

ica IV

IA-P

o-18

1

Bacteria subclade

Kang

iella

kor

eens

is D

SM 1

6069

Pseudoxa

nthomonas spadix

BD-a59

Thio

alka

limic

robi

um c

yclic

um A

LM1

Erwinia sp Ejp617

Tistrella mobilis KA081020-065

Shewanella halifaxensis HAW-EB4

Azospirillum brasilense Sp245

Soran

gium

cel

lulo

sum

So

ce56

Tere

dini

bact

er tu

rner

ae T

7901

Stigm

atel

la a

uran

tiaca

DW

43-1

Sphingopyxis alaskensis RB2256

Rickettsia rhipicephali str 3-7-fem

ale6-CW

PP

Desulfo

vibrio

gigas DSM 1382 = ATCC 19364

Acidiphilium cryptum JF-5

Bordetella pertussis 18323

Burkholderia vietnamiensis G4

Cam

pylo

bact

er la

ri R

M21

00

Aer

omon

as h

ydro

phila

sub

sp h

ydro

phila

ATC

C 7

966

Bacteria subclade

Hel

icob

acte

r ci

naed

i PA

GU

611

Psychrobacter cryohalolentis K5

Vibrio coralliilyticus

Chrom

ohal

obac

ter s

alex

igen

s DSM

304

3

Methylovorus glucosetrophus SIP3-4

Hyphom

icrobium nitrativorans N

L23

Enterobacter lignolyticus SCF1

Sulfuric

ella denitri

ficans s

kB26

Aliivibrio salmonicida LFI1238

Yersinia enterocolitica subsp enterocolitica 8081

Rhodoferax ferrireducens T118

Delftia acidovorans SPH-1

Burkholderia sp CCGE1001

Fran

cise

lla s

p TX

0773

08

Delftia sp Cs1-4

Cyc

locl

astic

us s

p P1

Pseudomonas denitrificans ATCC 13867

Sac

char

opha

gus

degr

adan

s 2-

40

Burkholderia lata

Bra

dyrh

izob

ium

olig

otro

phic

um S

58

Rho

dops

eudo

mon

as p

alus

tris

CG

A00

9 Brucella suis 1330

Magnetospirillum gryphiswaldense MSR-1

Phaeobacter gallaeciensis 210

Burkholderia sp CCGE1002

Proteus mirabilis HI4320

Brenneria sp EniD312

Methylobacterium

populi BJ001

Nitrospiraceae

Candidatus B

lochmannia chrom

aiodes str 640

Desulf

ovibr

io vu

lgaris

str H

ilden

boro

ugh

Caulobacter crescentus NA1000

Erwinia tasm

aniensis Et199

Xenorhabdus nematophila ATCC 19061

Spiribacte

r sp U

AH-SP71

Xanthomonas axonopodis pv citri str 3

06

Olig

otro

pha

carb

oxid

ovor

ans

OM

5

Candidatus H

odgkinia cicadicola Dsem

Novosphingobium pentaromativorans US6-1

Can

dida

tus

Libe

ribac

ter

sola

nace

arum

CLs

o-Z

C1

Salmonella enterica subsp enterica serovar Typhi str C

T18

Pelo

bact

er c

arbi

nolic

us D

SM 2

380

Desulf

ovibr

io ae

spoe

ensis

Asp

o-2

Polymorphum gilvum SL003B-26A1

Rickettsia sibirica 246

Vibrio vulnificus CMCP6

Escherichia coli O

83:H1 str N

RG

857C

Bar

tone

lla h

ense

lae

str

Hou

ston

-1

Shewanella frigidimarina NCIMB 400

Magnetococcus marinus MC-1

Shewanella woodyi ATCC 51908

Pseudomonas chlororaphis O6

Serratia sp AS12

Rickettsia australis str P

hillips

Proteus sp 3M

Pantoea ananatis LMG 20103

Idiomarina loihiensis L2TR

Shewanella sp W3-18-1

Geo

bact

er s

p M

18

Frederiksenia canicola

Wolbachia endosym

biont of Culex quinquefasciatus Pel

Wolbachia endosym

biont strain TRS

of Brugia m

alayi

Anaer

omyx

obac

ter d

ehal

ogen

ans

2CP-C

Collimonas fungivorans Ter331

Candidatus Kinetoplastibacterium blastocrithidii (ex Strigomonas culicis)

Bra

dyrh

izob

ium

sp

BTA

i1

Des

ulfu

rivib

rio a

lkal

iphi

lus

AH

T 2

Serratia plymuthica AS9

Geo

bact

er s

p M

21

Psychrobacter maritimus

Cupriavidus taiwanensis LMG 19424

Candidatus Kinetoplastibacterium galatii TCC219

Citrobacter rodentium ICC168

Bordetella pertussis Tohama I

Candidatus Photodesmus katoptron Akat1

Rickettsia prow

azekii str Madrid EB

rucella pinnipedialis B294

Klebsiella oxytoca KCTC 1686

Burkholderia thailandensis E264

Shewanella denitrificans OS217

Myx

ococ

cus

fulvu

s HW

-1

Des

ulfo

tale

a ps

ychr

ophi

la L

Sv5

4

Rickettsia felis U

RR

WX

Cal2

Cronobacter sakazakii ATCC BAA-894

Starkeya novella D

SM

506

Candidatus Kinetoplastibacterium desouzaii TCC079E

Salmonella enterica subsp enterica serovar Paratyphi A str ATC

C 9150

Rickettsia slovaca 13-BM

esorhizobium loti M

AF

F303099

Cel

lvib

rio ja

poni

cus

Ued

a107

Colwellia psychrerythraea 34H

Sphingobium chlorophenolicum L-1

Sul

furo

vum

sp

NB

C37

-1

Pectobacterium wasabiae WPP163

Thiocy

stis v

iolas

cens

DSM

198

Thioalkaliv

ibrio sp

K90mix

Pseudomonas aeruginosa PAO1

Dinoroseobacter shibae DFL 12 = DSM 16493

Haemophilus parainfluenzae T3T1

Sul

furo

spiri

llum

del

eyia

num

DS

M 6

946

Pseudoalteromonas atlantica T6c

Vibrio furnissii NCTC 11218

Gallibacterium anatis UMN179

Cam

pylo

bact

er h

omin

is A

TC

C B

AA

-381

Rahnella aquatilis CIP 7865 = ATCC 33071

Acinetobacter nosocomialis

Octadecabacter arcticus 238

Neorickettsia sennetsu str M

iyayama

Methylobacterium

radiotolerans JCM

2831

Pusillimonas sp T7-7

Moraxella catarrhalis RH4

Gallionella

capsif

erriform

ans ES-2

Salmonella enterica subsp enterica serovar Typhi str Ty2

Hyphomonas neptunium ATCC 15444

Jannaschia sp CCS1

Desulf

omicr

obium

bac

ulatu

m D

SM 4

028

Methylotenera versatilis 301

Rickettsia endosym

biont of Ixodes scapularis

Taylorella equigenitalis MCE9

Klebsiella pneumoniae subsp pneumoniae MGH 78578

Spiribacte

r salin

us M19-4

0

Anaplasma m

arginale str St Maries

Escherichia coli O

104:H4 str 2011C

-3493

Aci

dith

ioba

cillu

s fe

rriv

oran

s S

S3

Pseudomonas putida KT2440

Frateuria aurantia

DSM 6220

Bar

tone

lla q

uint

ana

str

Toul

ouse

Burkholderia sp CCGE1003

Lawso

nia in

trace

llular

is PHEM

N1-00

Actinobacillus succinogenes 130Z

Pseudomonas savastanoi pv phaseolicola 1448A

Rickettsia australis str C

utlack

Nitros

ococ

cus o

cean

i ATCC 1

9707

Paraglaciecola psychrophila 170

Mannheimia succiniciproducens MBEL55E

Methylobacterium

extorquens PA

1

Vibrio fischeri ES114

Dechloromonas aromatica RCB

Rickettsia canadensis str M

cKiel

Sphingomonas wittichii RW1

alpha proteobacterium HIMB59

Burkholderia pseudomallei K96243

Neisseria meningitidis MC58

Met

hylo

mic

robi

um a

lcal

iphi

lum

20Z

Hel

icob

acte

r fe

lis A

TC

C 4

9179

Agr

obac

teriu

m s

p H

13-3

Dic

tyog

lom

us tu

rgid

um D

SM

672

4

Xanthomonas arboric

ola pv pruni s

tr CFBP 5530

Cyc

locl

astic

us z

ancl

es 7

8-M

E

Can

dida

tus

Libe

ribac

ter

amer

ican

us s

tr S

ao P

aulo

Rhodobacter capsulatus SB 1003

Azospirillum sp B510

Aci

dith

ioba

cillu

s ca

ldus

SM

-1

Halorh

odos

pira h

aloph

ila S

L1

Hel

icob

acte

r m

uste

lae

1219

8

Rhodospirillum centenum SW

Pseudomonas protegens Pf-5

Pseudomonas synxantha BG33R

Psychrobacter sp PRwf-1

Candidatus Pelagibacter sp IMCC9063

Met

hylo

mon

as m

etha

nica

MC

09

Bar

tone

lla s

p T

T01

05

Variovorax paradoxus S110

Komagataeibacter medellinensis NBRC 3288

Syn

troph

obac

ter f

umar

oxid

ans

MP

OB

Aromatoleum aromaticum EbN1

Acidovorax avenae subsp avenae ATCC 19860

Xylella

fasti

diosa 9a5c

Shewanella putrefaciens CN-32

Bra

dyrh

izob

ium

sp

OR

S 2

78

Marinobacter sp BSs20148

Mannheimia haemolytica USDA-ARS-USMARC-185

Cam

pylo

bact

er fe

tus

subs

p te

stud

inum

03-

427

Klebsiella pneumoniae subsp pneumoniae HS11286

Escherichia coli U

MN

026

Taylorella asinigenitalis MCE3

Bar

tone

lla a

ustr

alis

Aus

tNH

1

Burkholderia cenocepacia HI2424

Pseudomonas entomophila L48

Sin

orhi

zobi

um s

p M

14

Enterobacter asburiae LF7a

Pasteurella multocida subsp multocida str Pm70

Legi

onel

la p

neum

ophi

la s

ubsp

pne

umop

hila

str

Phi

lade

lphi

a 1

Nitroso

monas europaea ATCC 19718

Ruegeria sp TM1040

Anaplasma centrale str Israel

Des

ulfo

bact

eriu

m a

utot

roph

icum

HR

M2

Herminiimonas arsenicoxydans

Laribacter hongkongensis HLHK9

Libe

ribac

ter

cres

cens

BT-

1

Alloch

rom

atium

vino

sum

DSM

180

Nitr

obac

ter

win

ogra

dsky

i Nb-

255

Burkholderia ambifaria AMMD

Methylibium petroleiphilum PM1

Magnetospirillum magneticum AMB-1

Nitroso

spira

multif

ormis A

TCC 25196

Methylotenera mobilis JLW8

Escherichia coli IA

I39

Candidatus Pelagibacter ubique HTCC1062

Candidatus Nasuia deltocephalinicola str NAS-ALF

Pelagibacterium

halotolerans B2

Psychromonas ingrahamii 37

Vibrio harveyi

Dickeya paradisiaca NCPPB 2511

Rickettsia m

assiliae MTU

5

Pseudoalteromonas haloplanktis TAC125

Rhodom

icrobium vannielii A

TC

C 17100

Halia

ngiu

m o

chra

ceum

DSM

143

65

Marinobacter adhaerens HP15

Desulf

ovibr

io sa

lexige

ns D

SM 2638

Pelo

bact

er p

ropi

onic

us D

SM 2

379

Janthinobacterium sp Marseille

Candi

datu

s Car

sone

lla ru

ddii P

V

Caldisericum

exile AZ

M16c01

Sul

furim

onas

aut

otro

phic

a D

SM

162

94

Des

ulfa

rcul

us b

aars

ii D

SM

207

5

Gayadomonas joobiniege G7

Acidobacteria

Bar

tone

lla tr

iboc

orum

CIP

105

476

Phaeobacter inhibens DSM 17395

Nitr

atiru

ptor

sp

SB

155-

2

Shigella sp LN126

alpha proteobacterium HIMB5

Granulibacter bethesdensis CGDNIH1

Fran

cise

lla tu

lare

nsis

sub

sp n

ovic

ida

U11

2

Met

hylo

mic

robi

um a

lbum

BG

8

Mar

inom

onas

med

iterra

nea

MM

B-1

Hal

obac

terio

vora

x m

arin

us S

J

Agr

obac

teriu

m r

adio

bact

er K

84

Pantoea sp At-9b

Comamonas sp 7D-2

Elusim

icrobium m

inutum P

ei191

Geo

bact

er d

alto

nii F

RC

-32

Escherichia coli O

157:H7 str S

akai

Serratia proteamaculans 568

Citrobacter koseri ATCC BAA-895

Burkholderia phenoliruptrix BR3459a

Fran

cise

lla p

hilo

mira

gia

subs

p ph

ilom

iragi

a AT

CC

250

17

Xanthomonas fuscans subsp fu

scans

Sin

orhi

zobi

um m

edic

ae W

SM

419

Rickettsia conorii str M

alish 7

Rickettsia peacockii str R

ustic

Rhi

zobi

um tr

opic

i CIA

T 8

99

Erwinia piriflorinigrans C

FBP 5888

Con

greg

ibac

ter l

itora

lis K

T71

Shewanella sediminis HAW-EB3

Edwardsiella ictaluri 93-146

Sideroxydans l

ithotro

phicus E

S-1Des

ulfov

ibrio

afric

anus

str W

alvis

Bay

Candidatus Tremblaya phenacola PAVE

Geo

bact

er u

rani

iredu

cens

Rf4

Spirochaetia

Pseudomonas monteilii SB3101

Mesorhizobium

australicum W

SM

2073

Thioalkaliv

ibrio su

lfidiphilu

s HL-E

bGr7

Rubrivivax gelatinosus IL144

Nitros

ococ

cus h

aloph

ilus N

c 4

Orientia tsutsugam

ushi str Boryong

Def

errib

acte

race

ae

Acinetobacter pittii PHEA-2

Shewanella pealeana ATCC 700345

Des

ulfo

bacc

a ac

etox

idan

s D

SM

111

09

Bordetella avium 197N

Vibrio cincinnatiensis

Acidiphilium multivorum AIU301

Yersinia pestis Pestoides F

Wolbachia endosym

biont of Onchocerca ochengi

Bra

dyrh

izob

ium

dia

zoef

ficie

ns U

SD

A 1

10

Burkholderiales bacterium JOSHI_001

Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis

Nitr

atifr

acto

r sa

lsug

inis

DS

M 1

6511

Brucella m

elitensis bv 1 str 16M

Wol

inel

la s

ucci

noge

nes

DS

M 1

740

Hirschia baltica ATCC 49814

Des

ulfo

bact

er p

ostg

atei

2ac

9

Maricaulis maris MCS10

Nau

tilia

pro

fund

icol

a A

mH

Pseudomonas brassicacearum subsp brassicacearum NFM421

Sphingobium sp SYK-6Haemophilus somnus 129PT

Desulfurispirillum

indicum S

5

Psychrobacter arcticus 273-4

Neisseria gonorrhoeae FA 1090

Yersinia pseudotuberculosis IP 32953

Comamonas testosteroni CNB-2

Novosphingobium sp PP1Y

Octadecabacter antarcticus 307

Can

dida

tus

Rut

hia

mag

nific

a st

r Cm

(Cal

ypto

gena

mag

nific

a) Rickettsia africae E

SF-5

Bacteria subclade

Ehrlichia canis str JakeC

andidatus Midichloria m

itochondrii IricVA

Brevundimonas subvibrioides ATCC 15264

Arc

obac

ter

nitr

ofig

ilis

DS

M 7

299

Klebsiella variicola At-22

Rhodospirillum rubrum F11

Polynucleobacter necessarius subsp asymbioticus QLW-P1DMWA-1

Vibrio parahaemolyticus RIMD 2210633

Thiomonas arsenitoxydans

Actinobacillus suis H91-0380

Parvularcula bermudensis HTCC2503

Legi

onel

la lo

ngbe

acha

e N

SW

150

Yersinia pestis KIM

10+

Aggregatibacter aphrophilus NJ8700

Psychromonas sp CNPT3

Brucella m

icroti CC

M 4915

Rhodobacter sphaeroides 241

Cupriavidus metallidurans CH34

Dickeya zeae Ech1591

Buchnera aphidicola str APS (Acyrthosiphon pisum)

Enterobacter aerogenes KCTC 2190

Desulfo

vibrio

desulfu

ricans N

D132

Halom

onas

elo

ngat

a DSM

258

1

Sin

orhi

zobi

um m

elilo

ti 10

21

Pandoraea sp RB-44

Gluconobacter oxydans 621H

Rickettsia helvetica C

9P9

Shewanella sp ANA-3

Serratia liquefaciens ATCC 27592

Rhi

zobi

um e

tli C

FN

42

Bac

teria

sub

clad

e

Pseudomonas syringae pv syringae B728a

Des

ulfo

bacu

la to

luol

ica

Tol2

Burkholderia mallei ATCC 23344

Shigella flexneri 2a str 301

Pseudomonas poae RE*1-1-14

Dechlorosoma suillum PS

Pseudomonas fluorescens Pf0-1

Rickettsia m

ontanensis str OS

U 85-930

Sodalis glossinidius str morsitans

Candidatus Tremblaya princeps PCIT

gam

ma

prot

eoba

cter

ium

HdN

1

Bordetella parapertussis Bpp5

Haemophilus influenzae Rd KW20

Mesorhizobium

opportunistum W

SM

2075

Alteromonas mediterranea

Thiobacillus d

enitrific

ans ATCC 25259

Bac

teria

sub

clad

e

Hel

icob

acte

r ac

inon

ychi

s st

r S

heeb

a

Acidovorax sp JS42

Enterobacter cloacae subsp cloacae ATCC 13047

Can

dida

tus

Vesi

com

yoso

cius

oku

tani

i HA

Nitroso

monas sp Is

79A3

Des

ulfa

tibac

illum

alk

eniv

oran

s AK

-01

Can

dida

tus

Libe

ribac

ter

asia

ticus

str

psy

62M

ethylobacterium extorquens C

M4

Aer

omon

as d

hake

nsis

AA

K1

Tolu

mon

as a

uens

is D

SM

918

7

Alca

nivo

rax

dies

elol

ei B

5

Hip

pea

mar

itim

a D

SM

104

11

Thiofla

vicoc

cus m

obilis

832

1

Paracoccus marcusii

Rhodospirillum rubrum ATCC 11170

Vibrio antiquarius

Yersinia frederiksenii

Hel

icob

acte

r he

patic

us A

TC

C 5

1449

Rahnella sp Y9602

Acinetobacter baumannii ATCC 17978

Leisingera methylohalidivorans DSM 14336

Shigella dysenteriae Sd197

Shewanella oneidensis MR-1

Vibrio anguillarum 775

Azorhizobium

caulinodans OR

S 571

Bordetella bronchiseptica 253

Candidatus Endolissoclinum faulkneri L2

Des

ulfo

caps

a su

lfexi

gens

DS

M 1

0523

Burkholderia rhizoxinica HKI 454

Pectobacterium carotovorum subsp carotovorum PC1

Agr

obac

teriu

m v

itis

S4

Neorickettsia risticii str Illinois

Rickettsia rickettsii str Iow

a

secondary endosymbiont of C

tenarytaina eucalypti

Bacteria subclade

Shewanella baltica OS223

Nitr

obac

ter

ham

burg

ensi

s X

14

Acinetobacter sp ADP1

Anaer

omyx

obac

ter s

p K

Sul

furic

urvu

m k

ujie

nse

DS

M 1

6994

Bar

tone

lla c

larr

idge

iae

73

Zymomonas mobilis subsp mobilis ZM4 = ATCC 31821

Xanthobacter autotrophicus P

y2

Enterobacter sp 638

Marinobacter hydrocarbonoclasticus VT8

Shewanella loihica PV-4

Micavibrio aeruginosavorus ARL-13

Ehrlichia muris AS145

Fran

cise

lla tu

lare

nsis

sub

sp tu

lare

nsis

SC

HU

S4

Desulf

ovibr

io hy

drot

herm

alis A

M13

= D

SM 1

4728

Candidatus Moranella endobia PCIT

Azospirillum lipoferum 4B

Candidatus Zinderia insecticola CARI

Met

hylo

cocc

us c

apsu

latu

s st

r Bat

h

Nitroso

monas sp AL212

Neisseria lactamica 020-06

Arc

obac

ter

sp L

Nitroso

cocc

us w

atson

ii C-11

3

Pseudomonas fulva 12-X

Bau

man

nia

cica

delli

nico

la s

tr H

c (H

omal

odis

ca c

oagu

lata

)

Fus

obac

teria

les

Candidatus B

lochmannia pennsylvanicus str B

PE

N

Wolbachia sp w

Ri

Burkholderia mallei SAVP1

Sul

furim

onas

den

itrifi

cans

DS

M 1

251

Alicycliphilus denitrificans BC

Rhi

zobi

um le

gum

inos

arum

bv

vici

ae 3

841

Ketogulonicigenium vulgare Y25

Pseudomonas stutzeri A1501

Coral

loco

ccus

cor

allo

ides

DSM

225

9

Ralstonia pickettii 12J

Rickettsia japonica Y

H

Anaplasma phagocytophilum

str HZ

Advenella kashmirensis WT001

Leptothrix cholodnii SP-6

Brucella abortus bv 1 str 9-941

Mar

inom

onas

sp

MW

YL1

Shewanella piezotolerans WP3

Azoarcus sp BH72

Acetobacter pasteurianus IFO 3283-01

Roseobacter denitrificans OCh 114

Haemophilus ducreyi 35000HP

Geo

bact

er m

etal

lired

ucen

s G

S-15

Myx

ococ

cus x

anth

us D

K 162

2

Phenylobacterium zucineum HLK1

Des

ulfo

cocc

us o

leov

oran

s H

xd3

Xanthomonas campestr

is pv c

ampestris

str ATCC 33913

Syn

ergi

stac

eae

Serratia symbiotica str Cinara cedri

Photorhabdus luminescens subsp laumondii TTO1

Anaer

omyx

obac

ter s

p Fw

109-

5

Bar

tone

lla b

acill

iform

is K

C58

3

Photobacterium profundum SS9

Pseudoxa

nthomonas suwonensis

11-1

Beijerinckia indica subsp indica A

TC

C 9039

Erwinia billingiae Eb661

Shewanella baltica OS155

Pseudomonas sp UW4

Edwardsiella tarda EIB202

Fran

cise

lla n

oatu

nens

is s

ubsp

orie

ntal

is s

tr To

ba 0

4

Methylobacterium

sp 4-46

Geo

bact

er b

emid

jiens

is B

em

Caulobacter segnis ATCC 21756

Bde

llovi

brio

bac

terio

voru

s st

r Tib

eriu

s

Pectobacterium atrosepticum SCRI1043

Hel

icob

acte

r pyl

ori 2

6695

Xanthomonas oryzae pv oryzae KACC 10331

Candidatus R

egiella insecticola LSR

1

Pseudomonas migulae

Vibrio cholerae O1 biovar El Tor str N16961

Sphingobium fuliginis ATCC 27551

Desulf

ohalo

bium

retb

aens

e DSM

569

2

Sphingobium japonicum UT26S

Hyphom

icrobium denitrificans A

TC

C 51888

Desulfo

vibrio

sp F

W1012B

Yersinia ruckeri

Cupriavidus necator N-1

Burkholderia glumae BGR1

Rickettsia typhi str W

ilmington

Archaea

Burkholderia multivorans ATCC 17616

Vibrio sp EJY3

Raoultella ornithinolytica B6

Thioalkaliv

ibrio nitra

tireduce

ns DSM 14787

Ferrimonas balearica DSM 9799

Pandoraea pnomenusa 3kgm

Halom

onas

sp

ZM3

The

rmod

esul

foba

cter

ium

geo

font

is O

PF

15

Verminephrobacter eiseniae EF01-2

Rickettsia heilongjiangensis 054

Ehrlichia ruminantium

str Welgevonden

Ehrlichia chaffeensis str Arkansas

Pseudomonas mendocina ymp

Xanthomonas horto

rum pv carotae st

r M081

Met

hylo

phag

a ni

tratir

educ

entic

resc

ens

Salmonella sp M

9397

Shigella sonnei Ss046

Sin

orhi

zobi

um fr

edii

NG

R23

4

Methylobacillus fla

gellatus KT

Gluconacetobacter diazotrophicus PA1 5

Dic

tyog

lom

us th

erm

ophi

lum

H-6

-12

Wolbachia endosym

biont of Drosophila m

elanogaster

Nitroso

monas eutro

pha C91

Actinobacillus pleuropneumoniae serovar 5b str L20

Dickeya dianthicola NCPPB 453

Bra

dyrh

izob

ium

japo

nicu

m U

SD

A 6

Candidatus Symbiobacter mobilis CR

Rickettsia parkeri str P

ortsmouth

Bar

tone

lla v

inso

nii s

ubsp

ber

khof

fii s

tr W

inni

e

Candidatus B

lochmannia floridanus

Ochrobactrum

anthropi AT

CC

49188

Glaciecola nitratireducens FR1064

Cam

pylo

bact

er je

juni

sub

sp je

juni

NC

TC

111

68 =

AT

CC

700

819

Thiomonas intermedia K12

Providencia sneebia DSM 19967

Xenorhabdus bovienii SS-2004

Herbaspirillum seropedicae SmR1

Burkholderia gladioli BSR3Burkholderia phymatum STM815

Novosphingobium aromaticivorans DSM 12444

uncultured Termite group 1 bacterium

phylotype Rs-D

17

Pararhodospirillum photometricum DSM 122

Paracoccus denitrificans PD1222

Rhodanobacter d

enitrific

ans

Candid

atus

Por

tiera

aley

rodid

arum

BT-

B-HRs

Salmonella enterica subsp enterica serovar Typhim

urium str LT2

Shewanella violacea DSS12

Desulfo

vibrio

alaskensis

G20

Hyphom

icrobium sp M

C1

Sulfitobacter sp DFL14

Pectobacterium sp SCC3193

Stenotrophomonas m

altophilia

K279a

Roseobacter litoralis Och 149

Mesorhizobium

ciceri biovar biserrulae WS

M1271

Methylobacterium

nodulans OR

S 2060

Ruegeria pomeroyi DSS-3

Candidatus Kinetoplastibacterium oncopeltii TCC290E

Achromobacter xylosoxidans A8

Burkholderia xenovorans LB400

[Pseudomonas syringae] pv tomato str DC3000

Alcani

vora

x bo

rkum

ensis

SK2

Pseudogulbenkiania sp NH8B

Shewanella baltica OS185

Xanthomonas albilineans GPE PC73

Polaromonas naphthalenivorans CJ2

Azotobacter vinelandii DJ

Parvibaculum

lavamentivorans D

S-1

Ralstonia eutropha JMP134

Cam

pylo

bact

er c

urvu

s 52

592

Des

ulfo

mon

ile ti

edje

i DS

M 6

799

Chelativorans sp B

NC

1

Erwinia pyrifoliae Ep196

Haloth

iobac

illus n

eapo

litanu

s c2

Dickeya dadantii Ech703

Vibrio tasmaniensis LGP32

Asticcacaulis excentricus CB 48B

acte

ria s

ubcl

ade

Ramlibacter tataouinensis TTB310

Aer

omon

as s

alm

onic

ida

subs

p sa

lmon

icid

a A

449

Methylovorus sp MP688

Pseudovibrio sp FO-BEG1

Photorhabdus asymbiotica

Thal

asso

lituus

ole

ivora

ns M

IL-1

Polaromonas sp JS666

Pantoea vagans C9-1

Des

ulfo

bulb

us p

ropi

onic

us D

SM

203

2

Yersinia pseudotuberculosis PB

1+

Cronobacter turicensis z3032

Wolbachia endosym

biont of Drosophila sim

ulans wH

a

Ketogulonicigenium vulgare W

SH-001

Aer

omon

as v

eron

ii B

565

Thauera sp MZ1T

Sul

furim

onas

got

land

ica

GD

1

Shimwellia blattae DSM 4481 = NBRC 105725

Sim

idui

a ag

ariv

oran

s S

A1

= D

SM

216

79

Syn

troph

us a

cidi

troph

icus

SB

Komagataeibacter hansenii ATCC 23769

Acinetobacter bereziniae

Methylocella silvestris B

L2

Ralstonia solanacearum GMI1000

Rickettsia bellii R

ML369-C

Candidatus H

amiltonella defensa 5AT (A

cyrthosiphon pisum)

Rickettsia akari str H

artford

Aggregatibacter actinomycetemcomitans D11S-1

Agr

obac

teriu

m fa

brum

str

C58

Salmonella bongori N

CTC

12419

Brucella canis A

TC

C 23365

Acinetobacter lwoffii

Burkholderia phytofirmans PsJN

Escherichia coli str K

-12 substr MG

1655

Brucella ovis A

TC

C 25840

Vibrio campbellii ATCC BAA-1116

Yersinia pestis CO

92

Oce

anim

onas

sp

GK

1

Brucella ceti T

E10759-12

Caulobacter sp K31

Cam

pylo

bact

er c

onci

sus

1382

6

Rahnella aquatilis HX2

Candidatus B

lochmannia vafer str B

VAF

secondary endosymbiont of H

eteropsylla cubana

Glaciecola sp 4H-3-7+YE-5

Desulfo

vibrio

desulfu

ricans s

ubsp desu

lfuric

ans str A

TCC 27774

Bra

dyrh

izob

iace

ae b

acte

rium

SG

-6C

Xanthomonas axo

nopodis pv c

itrumelo F1

Bde

llovi

brio

exo

voru

s JS

S

Rahnella genomosp 3

Methylocystis sp S

C2

Pseudoalteromonas sp SM9913

Hel

icob

acte

r cet

orum

MIT

99-

5656

Bar

tone

lla g

raha

mii

as4a

up

Shigella boydii Sb227

Cam

pylo

bact

er fe

tus

subs

p fe

tus

82-4

0

Acidovorax citrulli AAC00-1

Erythrobacter litoralis HTCC2594

Acinetobacter oleivorans DR1

Sul

furo

spiri

llum

bar

nesi

i SE

S-3

Paracoccus haeundaensis

Cox

iella

bur

netii

RS

A 4

93

Candidatus Riesia pediculicola USDA

Acidovorax ebreus TPSY

Desulfo

vibrio

piezophilu

s C1TLV

30

Alkalili

mnicola

ehrlic

hii M

LHE-1

Paracoccus aestuarii

Moraxella macacae 0408225

Thio

mic

rosp

ira c

runo

gena

XC

L-2

Chromobacterium violaceum ATCC 12472

Sulfitobacter guttiformis

Candidatus Kinetoplastibacterium crithidii (ex Angomonas deanei ATCC 30255)

Haemophilus parasuis SH0165

Geo

bact

er lo

vley

i SZ

Arc

obac

ter

butz

leri

RM

4018

Desulf

ovibr

io m

agne

ticus

RS-1

Aci

dith

ioba

cillu

s fe

rroo

xida

ns A

TC

C 5

3993

Bordetella petrii DSM 12804

Hel

icob

acte

r biz

zoze

roni

i CIII

-1

Xanthomonas campestris pv vesicatoria

str 85-10

Hah

ella

che

juen

sis

KCTC

239

6

Myx

ococ

cus

stip

itatu

s DSM

146

75

Escherichia fergusonii ATCC

35469

Geo

bact

er s

ulfu

rredu

cens

PCA

Shewanella sp MR-7

Candidatus Accumulibacter phosphatis clade IIA str U

W-1

Erwinia am

ylovora ATCC

49946

Alteromonas sp SN2

Serratia plymuthica 4Rx13

Candidatus Puniceispirillum marinum IMCC1322

Shewanella sp MR-4

The

rmod

esul

fata

tor

indi

cus

DS

M 1

5286

Rickettsia philipii str 364D

Shewanella amazonensis SB2B

Dic

helo

bact

er n

odos

us V

CS

1703

A

AnmK

MupP

MurU

AmgK

MurQ

Enterobacteriaceae

Alteromonadales

Pseudomonadales

Xanthomonadales

Gamma-proteobacteria

Alpha-proteobacteria

Beta-proteobacteria

Deltaepsilon-proteobacteria

Burkholderiales

Neisseriales

Rickettsiales

Vibrionaceae

Pasteurellaceae

Chromatiales

Oceanospirillales

Aeromonadaceae

Acetobacteraceae

Francisella

Myxococcales

Rhodobacterales

Sinorhizobium genus

Pseudo-alteromonadales

MurU pathway

FIG 8 Phylogenetic tree showing AnmK, MupP, AmgK, and MurU protein occurrence and co-conservation. The phylogenetic tree shown was constructed withiTOL (55) and a diversity set of 1,773 strains. The names of the relevant bacterial classes, orders, or families are indicated. The presence of MupP or other PGrecycling enzymes (33) in a given species is indicated by the colored regions at the outer edge of the tree and the legend at the lower right.

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reliant on the MurQ pathway and de novo synthesis. On the basis of these results, weconclude that MupP is likely to be the missing MurNAc-6P phosphatase enzymepreviously predicted to be functioning in the MurU pathway (33). In support of thisdesignation, the Mayer group has biochemically characterized MupP from Pseudomo-nas putida (44). They report in a parallel study that MupP specifically hydrolyzesMurNAc-6P to MurNAc in vitro. What remains unclear is why the MurU pathwayconverts MurNAc-6P to MurNAc before the AmgK kinase adds a phosphate back toform MurNAc-1P. In theory, the conversion of MurNAc-6P to MurNAc-1P could easily becatalyzed in a single step by a sugar phosphomutase. We therefore speculate that theless efficient pathway involving MupP and the formation of unphosphorylated MurNAcis likely to have additional physiological roles beyond PG recycling. Further studies arerequired to determine if and why the production of a steady-state pool of MurNAcmight be beneficial for bacteria that utilize the MurU PG recycling pathway.

Mutants with the PG recycling enzyme AmpD or the PG remodeling factor DacBinactivated have previously been identified as ampC inducers (7, 13, 14). Defects ineither enzyme are thought to promote the accumulation in the cytoplasm ofanhMurNAc peptides, which convert AmpR to an activator of ampC expression. Ablockade in PG sugar recycling by the MurU pathway has not previously been impli-cated in ampC induction or elevated beta-lactam resistance. The mechanism by whichinactivation of the MurU pathway stimulates increased ampC expression is not known.However, it seems unlikely that the failure to recycle the MurNAc sugars would preventproper peptide cleavage from anhMurNAc peptides by AmpD such that the inducerswould accumulate appreciably to activate AmpR. Instead, we favor the idea thatinhibition of the MurU pathway reduces the steady-state level of UDP-MurNAc-pep5because of limitations in UDP-MurNAc production. Because UDP-MurNAc-pep5 com-petes with anhMurNAc-pep5 for binding to AmpR (35), decreased UDP-MurNAc-pep5levels would alter the repressor/activator ratio and allow basal levels of anhMurNAc-pep5 to associate with AmpR to activate ampC expression and promote beta-lactamresistance. Although additional experimentation is required to test this hypothesis, theFos hypersensitivity caused by inactivation of the MurU pathway is consistent with adefect in UDP-MurNAc production in mutant cells.

The identification of a new cell wall recycling factor by the PampC::lacZ reporterscreen validates the utility of this approach for uncovering novel players involved in themaintenance of cell wall homeostasis in P. aeruginosa and likely other Gram-negativebacteria. The screen reported here was not saturated, suggesting that additional PGbiogenesis factors will be discovered upon continued screening. The identification andcharacterization of such factors will add to our growing understanding of the mecha-nisms by which bacteria build and maintain their cell wall and help us identifyvulnerabilities in the process to exploit for antibiotic targeting.

MATERIALS AND METHODSMedia, bacterial strains, and plasmids. P. aeruginosa PAO1 cells were grown in LB (1% tryptone,

0.5% yeast extract, 0.5% NaCl). When necessary, the medium was supplemented with 1 mM IPTG(isopropyl-�-D-thiogalactopyranoside), 5% sucrose, or 50 �g/ml X-Gal. For plasmid maintenance orintegration, gentamicin (Gm) and Tet were used at a concentration of 50 �g/ml. For AmpC beta-lactamase induction, Fox was used at a concentration of 50 �g/ml. Unless otherwise indicated, antibioticsfor viability/sensitivity assays were used at 25 (Fos), 4 (Caz), or 25 (Ctx) �g/ml.

E. coli cells were grown in LB. When necessary, the medium was supplemented with 100 �M IPTG.Unless otherwise indicated, the antibiotic concentrations used for E. coli were 25 (chloramphenicol andkanamycin), 10 (Gm), and 2 (Fos) �g/ml. The bacterial strains and plasmids used in this study are listedin Tables S1 to S3 in the supplemental material. Detailed descriptions of the strain and plasmidconstruction procedures can be found in Text S1.

Viability assays. For viability assays with P. aeruginosa or E. coli, overnight cell cultures werenormalized to an optical density at 600 nm (OD600) of 0.05 and subjected to serial 10-fold dilution.Five-microliter volumes of the 10�1 through 10�6 dilutions were then spotted onto the indicated agarand incubated at 30°C (P. aeruginosa) or 37°C (E. coli) for ~24 h prior to imaging. Fos MICs was determinedby the broth microdilution method. Overnight cell cultures were normalized to an OD600 of 0.0005 in LBand different concentrations of Fos and grown for ~24 h at 30°C. The MIC was defined as the lowestconcentration that inhibited growth.

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Screening for mutants that induce ampC expression. P. aeruginosa strain CF263 (PAO1 [PampC::lacZ]) was transposon mutagenized by mating with the E. coli donor SM10(�pir) harboring marinertransposon delivery vector pIT2 (50). The transposon confers Tet resistance. Mating mixtures were platedon LB agar supplemented with Tet (50 �g/ml) to select for transposon mutants and nalidixic acid(25 �g/ml) to select against the E. coli donor. The resulting collection of colonies was resuspended in LBbroth and stored at �80°C. Dilutions of the library were plated on LB containing X-Gal (40 �g/ml) toidentify mutants with a constitutively active PampC::lacZ reporter. The screen was not saturated, asindicated by the absence of ampD mutants among the isolates identified. We are therefore continuingto mine the library for additional mutants that induce the PampC::lacZ reporter.

Mapping of transposon insertion sites. Transposon insertions were mapped by arbitrarily primedPCR (50). Transposon-chromosomal DNA junctions were amplified from mutant chromosomal DNA withprimers Rnd1-PA (5= GGCCACGCGTCGACTAGTACNNNNNNNNNNGATAT 3=) and LacZ211 (5= TGC GGGCCT CTT CGC TAT TA 3=). The resulting PCR was used for a second PCR with primers Rnd2-PA(5= GGCCACGCGTCGACTAGTAC 3=) and LacZ148 (5= GGG TAA CGC CAG GGT TTT CC 3=). The final PCRproduct was sequenced with transposon-specific primer LacZ-124L (5= CAG TCA CGA CGT TGT AAA ACGACC). The transposon-chromosomal DNA junction was identified in the sequencing reads by a nucleotideBLAST search (51) against the PAO1 genome (52).

�-Galactosidase assays. �-Galactosidase assays were performed at room temperature. Cells from100 �l of culture at an OD600 of 0.1 to 0.6 were lysed with 30 �l of chloroform and mixed with 700 �lof Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4 heptahydrate). Each reactionmixture then received 200 �l of o-nitrophenyl-�-D-galactopyranoside (4 mg/ml in 0.1 M KPO4, pH 7.0),and the reaction was timed. When a medium yellow color developed, the reaction was stopped with400 �l of 1 M Na2CO3. The OD420 of the supernatant was determined, and the units of activity werecalculated with the equation U � (OD420 � 1,000)/[OD660 · time (in minutes) · volume of culture (inmilliliters)].

AmpC beta-lactamase activity assay. AmpC activity was assessed by nitrocefin hydrolysis. Over-night bacterial cultures were subcultured 1:20 in 3 ml of LB and grown for 2 h at 30°C and 200 rpm.Cultures were split 1:1 in 2 ml of LB with or without 50 �g/ml (final concentration) Fox and incubatedfor an additional 1.5 h at 30°C and 200 rpm. Following incubation, 1 ml of culture was pelleted at2,300 � g for 5 min, washed once with 1 ml of 50 mM sodium phosphate buffer (pH 7.0), andresuspended in 1 ml of the same cold buffer. Samples were placed on ice and lysed at 4°C bysonication with a microprobe (Q800R2; QSonica, Newtown, CT). Sonicated samples were centrifugedat 12,000 � g for 5 min at 4°C, and supernatants were collected. The protein concentration wasdetermined with a Bradford assay (53) with bovine serum albumin (BSA) as the standard (G-Biosciences/Geno Technology Inc., Saint Louis, MO). Nitrocefin hydrolysis assays were performed with 96-well plates.Each reaction mixture had a final volume of 250 �l of 50 mM sodium phosphate buffer (pH 7.0)containing 10 �g of protein and 20 �g of nitrocefin (Thermo Fischer Scientific Oxoid, Waltham, MA).Nitrocefin hydrolysis was monitored by measuring the absorbance at 486 nm every 5 min for 2 h at 30°C.

Phylogenetic analysis. A phylogenetic tree showing the distribution of the MurU pathway proteinsand MurQ in a diverse set of 1,773 bacterial taxa was constructed. The amino acid sequences of all of themembers of the MurU pathway, AnmK, and MurQ were used as queries in a BLASTp search against theNCBI nonredundant database (54) with an E value cutoff of 10�26. A list of all of the taxa for whichsignificant BLAST results were found was then sorted. We used a complex and diverse set of 1,773bacterial taxa called representative genomes that is available on NCBI (ftp://ftp.ncbi.nlm.nih.gov/blast/db/, Representative_Genomes.00.tar.gz). The phylogenetic tree was constructed with PhyloT (http://phylot.biobyte.de/), and BLASTp results were plotted against the tree. The occurrence of a MupP proteinis indicated by red, that of MurU is indicated by green, that of Amgk is indicated by blue, that of AnmKis indicated by purple, and that of MurQ is indicated by yellow. The tree was visualized and annotatedwith iToL (http://itol.embl.de/) (55).

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/mBio

.00102-17.TEXT S1, PDF file, 0.2 MB.TABLE S1, PDF file, 0.05 MB.TABLE S2, PDF file, 0.1 MB.TABLE S3, PDF file, 0.1 MB.

ACKNOWLEDGMENTSWe thank all of the members of the Bernhardt and Rudner labs for advice and

helpful discussions. Special thanks to Christoph Mayer and his group for communicat-ing their results prior to publication and for coordinating the submission of manuscriptswith us. Special thanks to Stephen Lory and Simon Dove for help with P. aeruginosamethods and for providing strains and expert advice.

This work was supported by the National Institute of Allergy and Infectious Diseasesof the National Institutes of Health (R33 AI111713 and R01 AI083365). C.F. was sup-

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ported in part by a postdoctoral fellowship from the Swiss National Science Foundation(project P2GEP3_162073). The funders had no role in study design, data collection andinterpretation, or the decision to submit the work for publication.

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