Malic Enzyme and Malolactic Enzyme Pathways Are Functionally Linked but Independently Regulated in Lactobacillus casei BL23

ABSTRACTLactobacillus caseiis the only lactic acid bacterium in which two pathways forl-malate degradation have been described: the malolactic enzyme (MLE) and the malic enzyme (ME) pathways. Whereas the ME pathway enablesL. caseito grow onl-malate, MLE does not support growth. Themlegene cluster consists of three genes encoding MLE (mleS), the putativel-malate transporter MleT, and the putative regulator MleR. Themaegene cluster consists of four genes encoding ME (maeE), the putative transporter MaeP, and the two-component system MaeKR. Since both pathways compete for the same substrate, we sought to determine whether they are coordinately regulated and their role inl-malate utilization as a carbon source. Transcriptional analyses revealed that themleandmaegenes are independently regulated and showed that MleR acts as an activator and requires internalization ofl-malate to induce the expression ofmlegenes. Notwithstanding, bothl-malate transporters were required for maximall-malate uptake, although only anmleTmutation caused a growth defect onl-malate, indicating its crucial role inl-malate metabolism. However, inactivation of MLE resulted in higher growth rates and higher final optical densities onl-malate. The limited growth onl-malate of the wild-type strain was correlated to a rapid degradation of the availablel-malate tol-lactate, which cannot be further metabolized. Taken together, our results indicate thatL. caseil-malate metabolism is not optimized for utilization ofl-malate as a carbon source but for deacidification of the medium by conversion ofl-malate intol-lactate via MLE.

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Streptococcus pyogenes Malate Degradation Pathway Links pH Regulation and Virulence

Infection and Immunity ◽

10.1128/iai.02814-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1162-1171 ◽

Cited By ~ 10

Author(s):

Elyse Paluscio ◽

Michael G. Caparon

Keyword(s):

Carbon Source ◽

Streptococcus Pyogenes ◽

Malic Enzyme ◽

Ph Regulation ◽

Carbon Sources ◽

Degradation Pathway ◽

Content Type ◽

Ph Response ◽

Expression Of Genes ◽

Genes Encoding

The ability ofStreptococcus pyogenesto infect different niches within its human host most likely relies on its ability to utilize alternative carbon sources. In examining this question, we discovered that all sequencedS. pyogenesstrains possess the genes for the malic enzyme (ME) pathway, which allows malate to be used as a supplemental carbon source for growth. ME is comprised of four genes in two adjacent operons, with the regulatory two-component MaeKR required for expression of genes encoding a malate permease (maeP) and malic enzyme (maeE). Analysis of transcription indicated that expression ofmaePandmaeEis induced by both malate and low pH, and induction in response to both cues is dependent on the MaeK sensor kinase. Furthermore, bothmaePEandmaeKRare repressed by glucose, which occurs via a CcpA-independent mechanism. Additionally, malate utilization requires the PTS transporter EI enzyme (PtsI), as a PtsI–mutant fails to express the ME genes and is unable to utilize malate. Virulence of selected ME mutants was assessed in a murine model of soft tissue infection. MaeP–, MaeK–, and MaeR–mutants were attenuated for virulence, whereas a MaeE–mutant showed enhanced virulence compared to that of the wild type. Taken together, these data show that ME contributes toS. pyogenes' carbon source repertory, that malate utilization is a highly regulated process, and that a single regulator controls ME expression in response to diverse signals. Furthermore, malate uptake and utilization contribute to the adaptive pH response, and ME can influence the outcome of infection.

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Requirement of the Lactobacillus casei MaeKR Two-Component System for l-Malic Acid Utilization via a Malic Enzyme Pathway

Applied and Environmental Microbiology ◽

10.1128/aem.02145-09 ◽

2009 ◽

Vol 76 (1) ◽

pp. 84-95 ◽

Cited By ~ 49

Author(s):

José María Landete ◽

Luisa García-Haro ◽

Amalia Blasco ◽

Paloma Manzanares ◽

Carmen Berbegal ◽

...

Keyword(s):

Malic Acid ◽

Malic Enzyme ◽

Lactobacillus Casei ◽

Response Regulator ◽

Two Component System ◽

Gene Encoding ◽

Streptococcus Uberis ◽

Dnase I Footprinting ◽

Malolactic Enzyme ◽

Two Component

ABSTRACT Lactobacillus casei can metabolize l-malic acid via malolactic enzyme (malolactic fermentation [MLF]) or malic enzyme (ME). Whereas utilization of l-malic acid via MLF does not support growth, the ME pathway enables L. casei to grow on l-malic acid. In this work, we have identified in the genomes of L. casei strains BL23 and ATCC 334 a cluster consisting of two diverging operons, maePE and maeKR, encoding a putative malate transporter (maeP), an ME (maeE), and a two-component (TC) system belonging to the citrate family (maeK and maeR). Homologous clusters were identified in Enterococcus faecalis, Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus uberis. Our results show that ME is essential for l-malic acid utilization in L. casei. Furthermore, deletion of either the gene encoding the histidine kinase or the response regulator of the TC system resulted in the loss of the ability to grow on l-malic acid, thus indicating that the cognate TC system regulates and is essential for the expression of ME. Transcriptional analyses showed that expression of maeE is induced in the presence of l-malic acid and repressed by glucose, whereas TC system expression was induced by l-malic acid and was not repressed by glucose. DNase I footprinting analysis showed that MaeR binds specifically to a set of direct repeats [5′-TTATT(A/T)AA-3′] in the mae promoter region. The location of the repeats strongly suggests that MaeR activates the expression of the diverging operons maePE and maeKR where the first one is also subjected to carbon catabolite repression.

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Employing a Recombinant Strain of Advenella mimigardefordensis for Biotechnical Production of Homopolythioesters from 3,3′-Dithiodipropionic Acid

Applied and Environmental Microbiology ◽

10.1128/aem.00007-12 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3286-3297 ◽

Cited By ~ 17

Author(s):

Yongzhen Xia ◽

Jan Hendrik Wübbeler ◽

Qingsheng Qi ◽

Alexander Steinbüchel

Keyword(s):

Carbon Source ◽

Dry Weight ◽

Pha Synthase ◽

Mineral Salts ◽

Content Type ◽

Genes Encoding ◽

Polyhydroxyalkanoate Synthase ◽

Encoding Gene ◽

Different Origins ◽

Severe Growth

ABSTRACTAdvenella mimigardefordensisstrain DPN7Twas genetically modified to produce poly(3-mercaptopropionic acid) (PMP) homopolymer by exploiting the recently unraveled process of 3,3′-dithiodipropionic acid (DTDP) catabolism. Production was achieved by systematically engineering the metabolism of this strain as follows: (i) deletion of its inherent 3MP dioxygenase-encoding gene (mdo), (ii) introduction of thebuk-ptboperon (genes encoding the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, fromClostridium acetobutylicum), and (iii) overexpression of its own polyhydroxyalkanoate synthase (phaCAm). These measures yielded the potent PMP production strainA. mimigardefordensisstrain SHX22. The deletion ofmdowas required for adequate synthesis of PMP due to the resulting accumulation of 3MP during utilization of DTDP. Overexpression of the plasmid-bornebuk-ptboperon caused a severe growth repression. This effect was overcome by inserting this operon into the genome. Polyhydroxyalkanoate (PHA) synthases from different origins were compared. The native PHA synthase ofA. mimigardefordensis(phaCAm) was obviously the best choice to establish homopolythioester production in this strain. In addition, the cultivation conditions, including an appropriate provision of the carbon source, were further optimized to enhance PMP production. The engineered strain accumulated PMP up to approximately 25% (wt/wt) of the cell dry weight when cultivated in mineral salts medium containing glycerol as the carbon source in addition to DTDP as the sulfur-providing precursor. According to our knowledge, this is the first report of PMP homopolymer production by a metabolically engineered bacterium using DTDP, which is nontoxic, as the precursor substrate.

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The Hydroxyquinol Degradation Pathway in Rhodococcus jostii RHA1 and Agrobacterium Species Is an Alternative Pathway for Degradation of Protocatechuic Acid and Lignin Fragments

Applied and Environmental Microbiology ◽

10.1128/aem.01561-20 ◽

2020 ◽

Vol 86 (19) ◽

Author(s):

Edward M. Spence ◽

Heather T. Scott ◽

Louison Dumond ◽

Leonides Calvo-Bado ◽

Sabrina di Monaco ◽

...

Keyword(s):

Gene Cluster ◽

Protocatechuic Acid ◽

Alternative Pathway ◽

Degradation Pathway ◽

Oxidative Decarboxylation ◽

Content Type ◽

Genes Encoding ◽

Rhodococcus Jostii Rha1 ◽

Similar Gene ◽

Rhodococcus Jostii

ABSTRACT Deletion of the pcaHG genes, encoding protocatechuate 3,4-dioxygenase in Rhodococcus jostii RHA1, gives a gene deletion strain still able to grow on protocatechuic acid as the sole carbon source, indicating a second degradation pathway for protocatechuic acid. Metabolite analysis of wild-type R. jostii RHA1 grown on medium containing vanillin or protocatechuic acid indicated the formation of hydroxyquinol (benzene-1,2,4-triol) as a downstream product. Gene cluster ro01857-ro01860 in Rhodococcus jostii RHA1 contains genes encoding hydroxyquinol 1,2-dioxygenase and maleylacetate reductase for degradation of hydroxyquinol but also putative mono-oxygenase (ro01860) and putative decarboxylase (ro01859) genes, and a similar gene cluster is found in the genome of lignin-degrading Agrobacterium species. Recombinant R. jostii mono-oxygenase and decarboxylase enzymes in combination were found to convert protocatechuic acid to hydroxyquinol. Hence, an alternative pathway for degradation of protocatechuic acid via oxidative decarboxylation to hydroxyquinol is proposed. IMPORTANCE There is a well-established paradigm for degradation of protocatechuic acid via the β-ketoadipate pathway in a range of soil bacteria. In this study, we have found the existence of a second pathway for degradation of protocatechuic acid in Rhodococcus jostii RHA1, via hydroxyquinol (benzene-1,2,4-triol), which establishes a metabolic link between protocatechuic acid and hydroxyquinol. The presence of this pathway in a lignin-degrading Agrobacterium sp. strain suggests the involvement of the hydroxyquinol pathway in the metabolism of degraded lignin fragments.

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Ethylene Glycol Metabolism in the Acetogen Acetobacterium woodii

Journal of Bacteriology ◽

10.1128/jb.00942-15 ◽

2016 ◽

Vol 198 (7) ◽

pp. 1058-1065 ◽

Cited By ~ 20

Author(s):

Dragan Trifunović ◽

Kai Schuchmann ◽

Volker Müller

Keyword(s):

Ethylene Glycol ◽

Gene Cluster ◽

Dual Function ◽

Acetobacterium Woodii ◽

Acetyl Coa ◽

Terminal Electron Acceptor ◽

Content Type ◽

Bacterial Microcompartments ◽

Genes Encoding ◽

Acetogenic Bacterium

ABSTRACTThe acetogenic bacteriumAcetobacterium woodiiis able to grow by the oxidation of diols, such as 1,2-propanediol, 2,3-butanediol, or ethylene glycol. Recent analyses demonstrated fundamentally different ways for oxidation of 1,2-propanediol and 2,3-butanediol. Here, we analyzed the metabolism of ethylene glycol. Our data demonstrate that ethylene glycol is dehydrated to acetaldehyde, which is then disproportionated to ethanol and acetyl coenzyme A (acetyl-CoA). The latter is further converted to acetate, and this pathway is coupled to ATP formation by substrate-level phosphorylation. Apparently, the product ethanol is in part further oxidized and the reducing equivalents are recycled by reduction of CO2to acetate in the Wood-Ljungdahl pathway. Biochemical data as well as the results of protein synthesis analysis are consistent with the hypothesis that the propane diol dehydratase (PduCDE) and CoA-dependent propionaldehyde dehydrogenase (PduP) proteins, encoded by thepdugene cluster, also catalyze ethylene glycol dehydration to acetaldehyde and its CoA-dependent oxidation to acetyl-CoA. Moreover, genes encoding bacterial microcompartments as part of thepdugene cluster are also expressed during growth on ethylene glycol, arguing for a dual function of the Pdu microcompartment system.IMPORTANCEAcetogenic bacteria are characterized by their ability to use CO2as a terminal electron acceptor by a specific pathway, the Wood-Ljungdahl pathway, enabling in most acetogens chemolithoautotrophic growth with H2and CO2. However, acetogens are very versatile and can use a wide variety of different substrates for growth. Here we report on the elucidation of the pathway for utilization of ethylene glycol by the model acetogenAcetobacterium woodii. This diol is degraded by dehydration to acetaldehyde followed by a disproportionation to acetate and ethanol. We present evidence that this pathway is catalyzed by the same enzyme system recently described for the utilization of 1,2-propanediol. The enzymes for ethylene glycol utilization seem to be encapsulated in protein compartments, known as bacterial microcompartments.

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Functional Analysis of the Lactobacillus casei BL23 Sortases

Applied and Environmental Microbiology ◽

10.1128/aem.02287-12 ◽

2012 ◽

Vol 78 (24) ◽

pp. 8684-8693 ◽

Cited By ~ 32

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.

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Deciphering a Marine Bone-Degrading Microbiome Reveals a Complex Community Effort

mSystems ◽

10.1128/msystems.01218-20 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Erik Borchert ◽

Antonio García-Moyano ◽

Sergio Sanchez-Carrillo ◽

Thomas G. Dahlgren ◽

Beate M. Slaby ◽

...

Keyword(s):

Gene Cluster ◽

Marine Environment ◽

Field Experiments ◽

Bone Matrix ◽

Taxonomic Diversity ◽

Bone Surface ◽

Metagenomic Sequence ◽

Content Type ◽

Bone Degradation ◽

Genes Encoding

ABSTRACT The marine bone biome is a complex assemblage of macro- and microorganisms; however, the enzymatic repertoire to access bone-derived nutrients remains unknown. The bone matrix is a composite material made up mainly of organic collagen and inorganic hydroxyapatite. We conducted field experiments to study microbial assemblages that can use organic bone components as nutrient source. Bovine and turkey bones were deposited at 69 m depth in a Norwegian fjord (Byfjorden, Bergen). Metagenomic sequence analysis was used to assess the functional potential of microbial assemblages from bone surface and the bone-eating worm Osedax mucofloris, which is a frequent colonizer of whale falls and known to degrade bone. The bone microbiome displayed a surprising taxonomic diversity revealed by the examination of 59 high-quality metagenome-assembled genomes from at least 23 bacterial families. Over 700 genes encoding enzymes from 12 relevant enzymatic families pertaining to collagenases, peptidases, and glycosidases putatively involved in bone degradation were identified. Metagenome-assembled genomes (MAGs) of the class Bacteroidia contained the most diverse gene repertoires. We postulate that demineralization of inorganic bone components is achieved by a timely succession of a closed sulfur biogeochemical cycle between sulfur-oxidizing and sulfur-reducing bacteria, causing a drop in pH and subsequent enzymatic processing of organic components in the bone surface communities. An unusually large and novel collagen utilization gene cluster was retrieved from one genome belonging to the gammaproteobacterial genus Colwellia. IMPORTANCE Bones are an underexploited, yet potentially profitable feedstock for biotechnological advances and value chains, due to the sheer amounts of residues produced by the modern meat and poultry processing industry. In this metagenomic study, we decipher the microbial pathways and enzymes that we postulate to be involved in bone degradation in the marine environment. We here demonstrate the interplay between different bacterial community members, each supplying different enzymatic functions with the potential to cover an array of reactions relating to the degradation of bone matrix components. We identify and describe a novel gene cluster for collagen utilization, which is a key function in this unique environment. We propose that the interplay between the different microbial taxa is necessary to achieve the complex task of bone degradation in the marine environment.

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Examination of antimicrobial potential in natural isolates of lactobacillus casei/paracasei group

Genetika ◽

10.2298/gensr1203661t ◽

2012 ◽

Vol 44 (3) ◽

pp. 661-677 ◽

Cited By ~ 2

Author(s):

Maja Tolinacki ◽

Jelena Lozo ◽

Katarina Veljovic ◽

Milan Kojic ◽

Djordje Fira ◽

...

Keyword(s):

Gene Cluster ◽

Abc Transporter ◽

Lactobacillus Casei ◽

Bacteriocin Production ◽

Accessory Protein ◽

Terminal Sequence ◽

Antimicrobial Potential ◽

Genes Encoding ◽

Natural Isolates ◽

Production Strain

The aim of this study was to investigate the antimicrobial potential of 52 natural isolates of Lactobacillus casei/paracasei. The incidence of relevant genes encoding BacSJ (bacSJ2-8/bacSJ2-8i gene cluster), acidocin 8912 (acdT), ABC-transporter (abcT) and accessory protein (acc) was also studied. These genes were found to be widespread amongst the analyzed L. casei/paracasei strains. The bacSJ2-8/bacSJ2-8i gene cluster was present in 49 (94.23%) and acdT in 41 (78.85%) of the 52 tested strains. Forty of these strains (76.92%) harbored both analyzed genes. Interestingly, only 17 strains (32.69%) with the bacSJ2-8/bacSJ2-8i gene cluster and/or the acdT gene showed bacteriocin production. Strain L. paracasei BGNK1-62 contained the bacSJ2-8/bacSJ2-8i gene cluster, but did not produce bacteriocin BacSJ possibly due to absence of the abcT and acc genes. Hence, these genes were introduced into BGNK1-62 by transformation with constructed plasmid pA2A, after which BacSJ was produced. In addition, it was found that L. paracasei BGGR2-66 produced new bacteriocin designated as BacGR that was biochemically characterized and its N- terminal sequence was determined.

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Corynebacterium glutamicum Tailored for Efficient Isobutanol Production

Applied and Environmental Microbiology ◽

10.1128/aem.02972-10 ◽

2011 ◽

Vol 77 (10) ◽

pp. 3300-3310 ◽

Cited By ~ 224

Author(s):

Bastian Blombach ◽

Tanja Riester ◽

Stefan Wieschalka ◽

Christian Ziert ◽

Jung-Won Youn ◽

...

Keyword(s):

Alcohol Dehydrogenase ◽

Corynebacterium Glutamicum ◽

Malic Enzyme ◽

Pyruvate Dehydrogenase Complex ◽

Specific Yield ◽

Acetohydroxyacid Synthase ◽

Content Type ◽

Production Phase ◽

Genes Encoding ◽

Alcohol Dehydrogenase 2

ABSTRACTWe recently engineeredCorynebacterium glutamicumfor aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of theilvBNCDgenes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineeredC. glutamicumfor the production of isobutanol from glucose under oxygen deprivation conditions by inactivation ofl-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase fromLactococcus lactis, alcohol dehydrogenase 2 (ADH2) fromSaccharomyces cerevisiae, and expression of thepntABtranshydrogenase genes fromEscherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (YP/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 fromS. cerevisiaewas involved in isobutanol formation withC. glutamicum, and overexpression of the correspondingadhAgene increased the YP/Sto 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the YP/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H+to NADPH+H+. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain,C. glutamicumΔaceEΔpqoΔilvEΔldhAΔmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h−1, and showed an overall YP/Sof about 0.48 mol per mol of glucose in the production phase.

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The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02413-15 ◽

2015 ◽

Vol 60 (2) ◽

pp. 1085-1090 ◽

Cited By ~ 12

Author(s):

Jennifer Nowak ◽

Thamarai Schneiders ◽

Harald Seifert ◽

Paul G. Higgins

Keyword(s):

Acinetobacter Baumannii ◽

Antimicrobial Susceptibility ◽

Efflux Pump ◽

Response Regulator ◽

Two Component System ◽

Content Type ◽

Susceptibility Data ◽

Genes Encoding ◽

Induced Changes

ABSTRACTOverexpression of the resistance-nodulation-cell division-type efflux pump AdeABC is often associated with multidrug resistance inAcinetobacter baumanniiand has been linked to mutations in the genes encoding the AdeRS two-component system. In a previous study, we reported that the Asp20→Asn amino acid substitution in the response regulator AdeR is associated withadeBoverexpression and reduced susceptibility to the antimicrobials levofloxacin, tigecycline, and trimethoprim-sulfamethoxazole. To further characterize the effect of the Asp20→Asn substitution on antimicrobial susceptibility, the expression of the efflux genesadeB,adeJ, andadeG, and substrate accumulation, four plasmid constructs [containingadeR(Asp20)S,adeR(Asn20)S,adeR(Asp20)SABC, andadeR(Asn20)SABC] were introduced into theadeRSABC-deficientA. baumanniiisolate NIPH 60. NeitheradeRSconstruct induced changes in antimicrobial susceptibility or substrate accumulation from that for the vector-only control. TheadeR(Asp20)SABCtransformant showed reduced susceptibility to 6 antimicrobials and accumulated 12% less ethidium than the control, whereas the Asn20 variant showed reduced susceptibility to 6 of 8 antimicrobial classes tested, and its ethidium accumulation was only 72% of that observed for the vector-only construct.adeBexpression was 7-fold higher in theadeR(Asn20)SABCtransformant than in its Asp20 variant. No changes inadeGoradeJexpression or in acriflavine or rhodamine 6G accumulation were detected. The antimicrobial susceptibility data suggest that AdeRS does not regulate any resistance determinants other than AdeABC. Furthermore, the characterization of the Asp20→Asn20 substitution proves that the reduced antimicrobial susceptibility previously associated with this substitution was indeed caused by enhanced efflux activity of AdeB.

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