scholarly journals Functional Analysis of the Lactobacillus casei BL23 Sortases

2012 ◽  
Vol 78 (24) ◽  
pp. 8684-8693 ◽  
Author(s):  
Diego Muñoz-Provencio ◽  
Jesús Rodríguez-Díaz ◽  
María Carmen Collado ◽  
Philippe Langella ◽  
Luis G. Bermúdez-Humarán ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lactobacillus Casei ◽  
Double Mutant ◽  
Mutant Cell ◽  
Surface Hydrophobicity ◽  
Surface Proteins ◽  
Reporter Protein ◽  
Content Type ◽  
Genes Encoding ◽  
Class C

ABSTRACTSortases are a class of enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria.Lactobacillus caseiBL23 harbors four sortase genes, two belonging to class A (srtA1andsrtA2) and two belonging to class C (srtC1andsrtC2). Class C sortases were clustered with genes encoding their putative substrates that were homologous to the SpaEFG and SpaCBA proteins that encode mucus adhesive pili inLactobacillus rhamnosusGG. Twenty-three genes encoding putative sortase substrates were identified in theL. caseiBL23 genome with unknown (35%), enzymatic (30%), or adhesion-related (35%) functions. Strains disrupted insrtA1,srtA2,srtC1, andsrtC2and ansrtA1 srtA2double mutant were constructed. The transcription of all four sortase encoding genes was detected, but only the mutation ofsrtA1resulted in a decrease in bacterial surface hydrophobicity. The β-N-acetyl-glucosaminidase and cell wall proteinase activities of whole cells diminished in thesrtA1mutant and, to a greater extent, in thesrtA1 srtA2double mutant. Cell wall anchoring of the staphylococcal NucA reporter protein fused to a cell wall sorting sequence was also affected in thesrtAmutants, and the percentages of adhesion to Caco-2 and HT-29 intestinal epithelial cells were reduced for thesrtA1 srtA2strain. Mutations insrtC1orsrtC2result in an undetectable phenotype. Together, these results suggest that SrtA1 is the housekeeping sortase inL. caseiBL23 and SrtA2 would carry out redundant or complementary functions that become evident when SrtA1 activity is absent.

scholarly journals C-Terminal WxL Domain Mediates Cell Wall Binding in Enterococcus faecalis and Other Gram-Positive Bacteria

2006 ◽  
Vol 189 (4) ◽  
pp. 1244-1253 ◽  
Author(s):  
Sophie Brinster ◽  
Sylviane Furlan ◽  
Pascale Serror
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Enterococcus Faecalis ◽  
Surface Proteins ◽  
Reporter Protein ◽  
Gram Positive ◽  
Whole Cells ◽  
Gram Positive Bacteria ◽  
Cell Wall Binding ◽  
Genes Encoding

ABSTRACT Analysis of the genome sequence of Enterococcus faecalis clinical isolate V583 revealed novel genes encoding surface proteins. Twenty-seven of these proteins, annotated as having unknown functions, possess a putative N-terminal signal peptide and a conserved C-terminal region characterized by a novel conserved domain designated WxL. Proteins having similar characteristics were also detected in other low-G+C-content gram-positive bacteria. We hypothesized that the WxL region might be a determinant of bacterial cell location. This hypothesis was tested by generating protein fusions between the C-terminal regions of two WxL proteins in E. faecalis and a nuclease reporter protein. We demonstrated that the C-terminal regions of both proteins conferred a cell surface localization to the reporter fusions in E. faecalis. This localization was eliminated by introducing specific deletions into the domains. Interestingly, exogenously added protein fusions displayed binding to whole cells of various gram-positive bacteria. We also showed that the peptidoglycan was a binding ligand for WxL domain attachment to the cell surface and that neither proteins nor carbohydrates were necessary for binding. Based on our findings, we propose that the WxL region is a novel cell wall binding domain in E. faecalis and other gram-positive bacteria.

scholarly journals A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Shauna D. Drumm ◽  
Rebecca Owens ◽  
Jennifer Mitchell ◽  
Orla M. Keane
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Surface Proteins ◽  
Host Cells ◽  
Mass Spectrometry Analysis ◽  
In Vitro Assays ◽  
Immune Recognition ◽  
Independent Manner ◽  
Genes Encoding

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins. Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes. Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages. These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.

scholarly journals Helicobacter pylori Peptidoglycan Modifications Confer Lysozyme Resistance and Contribute to Survival in the Host

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
Ge Wang ◽  
Leja F. Lo ◽  
Lennart S. Forsberg ◽  
Robert J. Maier
Keyword(s):  
Cell Wall ◽  
Helicobacter Pylori ◽  
Double Mutant ◽  
Lytic Enzyme ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
H Pylori ◽  
Modification Enzyme

ABSTRACTThe prominent host muramidase lysozyme cleaves bacterial peptidoglycan (PG), and the enzyme is abundant in mucosal secretions. The lytic enzyme susceptibility of Gram-negative bacteria and mechanisms they use to thwart lytic enzyme activity are poorly studied. We previously characterized aHelicobacter pyloriPG modification enzyme, an N-deacetylase (PgdA) involved in lysozyme resistance. In this study, another PG modification enzyme, a putative PG O-acetyltransferase (PatA), was identified. Mass spectral analysis of the purified PG demonstrated that apatAstrain contained a greatly reduced amount of acetylated muropeptides, indicating a role for PatA inH. pyloriPG O-acetylation. The PG modification mutant strains (pgdA,patA, orpgdA patA) were more susceptible to lysozyme killing than the parent, but this assay required high lysozyme levels (up to 50 mg/ml). However, addition of host lactoferrin conferred lysozyme sensitivity toH. pylori, at physiologically relevant concentrations of both host components (3 mg/ml lactoferrin plus 0.3 mg/ml lysozyme). ThepgdA patAdouble mutant strain was far more susceptible to lysozyme/lactoferrin killing than the parent. Peptidoglycan purified from apgdA patAmutant was five times more sensitive to lysozyme than PG from the parent strain, while PG from both single mutants displayed intermediate sensitivity. Both sensitivity assays for whole cells and for purified PGs indicated that the modifications mediated by PgdA and PatA have a synergistic effect, conferring lysozyme tolerance. In a mouse infection model, significant colonization deficiency was observed for the double mutant at 3 weeks postinoculation. The results show that PG modifications affect the survival of a Gram-negative pathogen.IMPORTANCEPathogenic bacteria evade host antibacterial enzymes by a variety of mechanisms, which include resisting lytic enzymes abundant in the host. Enzymatic modifications to peptidoglycan (PG, the site of action of lysozyme) are a known mechanism used by Gram-positive bacteria to protect against host lysozyme attack. However, Gram-negative bacteria contain a thin layer of PG and a recalcitrant outer membrane permeability barrier to resist lysis, so molecular modifications to cell wall structure in order to combat lysis remain largely unstudied. Here we show that twoHelicobacter pyloriPG modification enzymes (PgdA and PatA) confer a clear protective advantage to a Gram-negative bacterium. They protect the bacterium from lytic enzyme degradation, albeit via different PG modification activities. Many pathogens are Gram negative, so some would be expected to have a similar cell wall-modifying strategy. Understanding such strategies may be useful for combating pathogen growth.

scholarly journals Five Genes Encoding Surface-Exposed LPXTG Proteins Are Enriched in Hospital-Adapted Enterococcus faecium Clonal Complex 17 Isolates

2007 ◽  
Vol 189 (22) ◽  
pp. 8321-8332 ◽  
Author(s):  
Antoni P. A. Hendrickx ◽  
Willem J. B. van Wamel ◽  
George Posthuma ◽  
Marc J. M. Bonten ◽  
Rob J. L. Willems
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecium ◽  
Clonal Complex ◽  
Mrna Level ◽  
Surface Protein ◽  
Surface Proteins ◽  
Dot Blot ◽  
Genes Encoding ◽  
Invasive Infections

ABSTRACT Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation.

scholarly journals Staphylococcus epidermidis MSCRAMM SesJ Is Encoded in Composite Islands

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Srishtee Arora ◽  
Xiqi Li ◽  
Andrew Hillhouse ◽  
Kranti Konganti ◽  
Sara V. Little ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Virulence Factors ◽  
Staphylococcus Epidermidis ◽  
Core Genome ◽  
Clinical Isolates ◽  
Modification System ◽  
Content Type ◽  
Genes Encoding ◽  
Staphylococcal Cassette Chromosome

ABSTRACT Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCCmec) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB, and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR_B or DR_E. When inserted via DR_E, the sesJ gene was encoded in the SCC element. When inserted via DR_B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates. IMPORTANCE S. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis, is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections.

scholarly journals Comparative RNA-seq-Based Transcriptome Analysis of the Virulence Characteristics of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius Strains Isolated from Small Animals

2015 ◽  
Vol 60 (2) ◽  
pp. 962-967 ◽  
Author(s):  
Natacha Couto ◽  
Adriana Belas ◽  
Manuela Oliveira ◽  
Paulo Almeida ◽  
Carla Clemente ◽  
...  
Keyword(s):  
Virulence Genes ◽  
Expression Profiles ◽  
Brain Heart Infusion ◽  
Surface Proteins ◽  
Rna Seq ◽  
Staphylococcus Pseudintermedius ◽  
Methicillin Resistant ◽  
Content Type ◽  
Genes Encoding

ABSTRACTStaphylococcus pseudintermediusis often associated with pyoderma, which can turn into a life-threatening disease. The dissemination of highly resistant isolates has occurred in the last 10 years and has challenged antimicrobial treatment of these infections considerably. We have compared the carriage of virulence genes and biofilm formation between methicillin-resistant and methicillin-susceptibleS. pseudintermedius(MRSP and MSSP, respectively) isolates and theirin vitrogene expression profiles by transcriptome sequencing (RNA-seq). Isolates were relatively unevenly distributed among the fouragrgroups, andagrtype III predominated in MRSP. Five virulence genes were detected in all isolates. Only thespsOgene was significantly associated with MSSP isolates (P= 0.04). All isolates produced biofilm in brain heart infusion broth (BHIB)–4% NaCl. MSSP isolates produced more biofilm on BHIB and BHIB–1% glucose media than MRSP isolates (P= 0.03 andP= 0.02, respectively). Virulence genes encoding surface proteins and toxins (spsA,spsB,spsD,spsK,spsL,spsN,nucC,coa, andluk-I) and also prophage genes (encoding phage capsid protein, phage infection protein, two phage portal proteins, and a phage-like protein) were highly expressed in the MRSP isolate (compared with the MSSP isolate), suggesting they may play a role in the rapid and widespread dissemination of MRSP. This study indicates that MRSP may upregulate surface proteins, which may increase the adherence of MRSP isolates (especially sequence type 71 [ST71]) to corneocytes. MSSP isolates may have an increased ability to form biofilm under acidic circumstances, through upregulation of the entirearcoperon. Complete understanding ofS. pseudintermediuspathogenesis and host-pathogen signal interaction during infections is critical for the treatment and prevention ofS. pseudintermediusinfections.

scholarly journals Direct Target Network of the Neurospora crassa Plant Cell Wall Deconstruction Regulators CLR-1, CLR-2, and XLR-1

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
James P. Craig ◽  
Samuel T. Coradetti ◽  
Trevor L. Starr ◽  
N. Louise Glass
Keyword(s):  
Gene Expression ◽  
Cell Wall ◽  
Transcription Factors ◽  
Neurospora Crassa ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Nutrient Sensing ◽  
Wall Material ◽  
Content Type ◽  
Genes Encoding

ABSTRACTFungal deconstruction of the plant cell requires a complex orchestration of a wide array of intracellular and extracellular enzymes. InNeurospora crassa, CLR-1, CLR-2, and XLR-1 have been identified as key transcription factors regulating plant cell wall degradation in response to soluble sugars. The XLR-1 regulon was defined using a constitutively active mutant allele, resulting in hemicellulase gene expression and secretion under noninducing conditions. To define genes directly regulated by CLR-1, CLR-2, and XLR-1, we performed chromatin immunoprecipitation and next-generation sequencing (ChIPseq) on epitope-tagged constructs of these three transcription factors. WhenN. crassais exposed to plant cell wall material, CLR-1, CLR-2, and XLR-1 individually bind to the promoters of the most strongly induced genes in their respective regulons. These include promoters of genes encoding cellulases for CLR-1 and CLR-2 (CLR-1/CLR-2) and promoters of genes encoding hemicellulases for XLR-1. CLR-1 bound to its regulon under noninducing conditions; however, this binding alone did not translate into gene expression and enzyme secretion. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest paralog inSaccharomyces cerevisiae, Gal4p. Coimmunoprecipitation studies showed that CLR-1 and CLR-2 act in a homocomplex but not as a CLR-1/CLR-2 heterocomplex.IMPORTANCEUnderstanding fungal regulation of complex plant cell wall deconstruction pathways in response to multiple environmental signals via interconnected transcriptional circuits provides insight into fungus/plant interactions and eukaryotic nutrient sensing. Coordinated optimization of these regulatory networks is likely required for optimal microbial enzyme production.

scholarly journals Cloning and Characterization of PRA1, a Gene Encoding a Novel pH-Regulated Antigen of Candida albicans

1998 ◽  
Vol 180 (2) ◽  
pp. 282-289 ◽  
Author(s):  
Maria Sentandreu ◽  
M. Victoria Elorza ◽  
Rafael Sentandreu ◽  
William A. Fonzi
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Immunoblot Analysis ◽  
Surface Proteins ◽  
Cell Fractionation ◽  
Expression Library ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Surface Localization ◽  
Opportunistic Fungal Pathogen

ABSTRACT Candida albicans is an opportunistic fungal pathogen of humans. The cell wall of the organism defines the interface between the pathogen and host tissues and is likely to play an essential and pivotal role in the host-pathogen interaction. The components of the cell wall critical to this interaction are undefined. Immunoscreening of a lambda expression library with sera raised against mycelial cell walls of C. albicans was used to identify genes encoding cell surface proteins. One of the positive clones represented a candidal gene that was differentially expressed in response to changes in the pH of the culture medium. Maximal expression occurred at neutral pH, with no expression detected below pH 6.0. On the basis of the expression pattern, the corresponding gene was designatedPRA1, for pH-regulated antigen. The protein predicted from the nucleotide sequence was 299 amino acids long with motifs characteristic of secreted glycoproteins. The predicted surface localization and N glycosylation of the protein were directly demonstrated by cell fractionation and immunoblot analysis. Deletion of the gene imparted a temperature-dependent defect in hypha formation, indicating a role in morphogenesis. The PRA1 protein was homologous to surface antigens of Aspergillus spp. which react with serum from aspergillosis patients, suggesting that thePRA1 protein may have a role in the host-parasite interaction during candidal infection.

scholarly journals Human IgM Inhibits the Formation of Titan-Like Cells in Cryptococcus neoformans

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Nuria Trevijano-Contador ◽  
Kaila M. Pianalto ◽  
Connie B. Nichols ◽  
Oscar Zaragoza ◽  
J. Andrew Alspaugh ◽  
...  
Keyword(s):  
Cell Wall ◽  
Stress Response ◽  
B Cell ◽  
Cryptococcus Neoformans ◽  
Cell Formation ◽  
Transcriptional Analysis ◽  
Content Type ◽  
Human Immunoglobulins ◽  
Genes Encoding

ABSTRACT Human studies have shown associations between cryptococcal meningitis and reduced IgM memory B cell levels, and studies in IgM- and/or B cell-deficient mice have demonstrated increased Cryptococcus neoformans dissemination from lungs to brain. Since immunoglobulins are part of the immune milieu that C. neoformans confronts in a human host, and its ability to form titan cells is an important virulence mechanism, we determined the effect of human immunoglobulins on C. neoformans titan cell formation in vitro. (i) Fluorescence microscopy showed normal human IgG and IgM bind C. neoformans. (ii) C. neoformans grown in titan cell-inducing medium with IgM, not IgG, inhibited titan-like cell formation. (iii) Absorption of IgM with laminarin or curdlan (branched and linear 1-3-beta-d-glucans, respectively) decreased this effect. (iv) Transmission electron microscopy revealed that cells grown with IgM had small capsules and unique features not seen with cells grown with IgG. (v) Comparative transcriptional analysis of cell wall, capsule, and stress response genes showed that C. neoformans grown with IgM, not IgG or phosphate-buffered saline (PBS), had decreased expression of chitin synthetase, CHS1, CHS2, and CHS8, and genes encoding cell wall carbohydrate synthetases α-1-3-glucan (AGS1) and β-1,3-glucan (FKS1). IgM also decreased expression of RIM101 and HOG1, genes encoding central regulators of C. neoformans stress response pathways and cell morphogenesis. Our data show human IgM affects C. neoformans morphology in vitro and suggest that the hypothesis that human immunoglobulins may affect C. neoformans virulence in vivo warrants further investigation.

scholarly journals Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of β-Lactamase-Producing Escherichia coli

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Kristin R. Baker ◽  
Helga Høeg Sigurðardóttir ◽  
Bimal Jana ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Lag Phase ◽  
Growth Curve Analysis ◽  
Wild Type ◽  
Content Type ◽  
Cell Wall Biogenesis ◽  
Genes Encoding ◽  
In The Wild ◽  
Nonessential Genes

ABSTRACT Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for “helper” drugs reversing cephem resistance in Escherichia coli strains producing β-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.

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