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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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