Regulation of the Gene for Alanine Racemase Modulates Amino Acid Metabolism with Consequent Alterations in Cell Wall Properties and Adhesive Capability in Brucella spp.

Brucella, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. Moreover, Brucella mutants with alr gene deletions (Δalr) exhibit potential as vaccine candidates. However, the mechanisms that underlie the detrimental effects of alr knockouts on Brucella pathogenicity remain elusive. Here, initially, we conducted a bioinformatics analysis of Alr, which demonstrated a high degree of conservation of the protein within Brucella spp. Subsequent metabolomics studies unveiled alterations in amino acid pathways following deletion of the alr gene. Furthermore, alr deletion in Brucella suis S2 induced decreased resistance to stress, antibiotics, and other factors. Transmission electron microscopy of simulated macrophage intracellular infection revealed damage to the cell wall in the Δalr strain, whereas propidium iodide staining and alkaline phosphatase and lactate dehydrogenase assays demonstrated alterations in cell membrane permeability. Changes in cell wall properties were revealed by measurements of cell surface hydrophobicity and zeta potential. Finally, the diminished adhesion capacity of the Δalr strain was shown by immunofluorescence and bacterial enumeration assays. In summary, our findings indicate that the alr gene that regulates amino acid metabolism in Brucella influences the properties of the cell wall, which modulates bacterial adherence capability. This study is the first demonstration that Alr impacts virulence by modulating bacterial metabolism, thereby providing novel insights into the pathogenic mechanisms of Brucella spp.

Computational and experimental analyses of alanine racemase suggest new avenues for developing allosteric small-molecule antibiotics.

Given the ever-present threat of antibacterial resistance, there is an urgent need to identify new antibacterial drugs and targets. One such target is alanine racemase (Alr), an enzyme required for bacterial cell-wall biosynthesis. Alr is an attractive drug target because it is essential for bacterial survival but is absent in humans. Existing drugs targeting Alr lack specificity and have severe side effects. We here investigate alternative mechanisms of Alr inhibition. Alr functions exclusively as an obligate homodimer, so we probed seven conserved interactions on the dimer interface, distant from the enzymatic active site, to identify possible allosteric influences on activity. Using the Alr from Mycobacterium tuberculosis (MT) as a model, we found that the Lys261/Asp135 salt bridge is critical for catalytic activity. The Lys261Ala mutation completely inactivated the enzyme, and the Asp135Ala mutation reduced catalytic activity eight-fold. Further investigation suggested a potential drug-binding site near the Lys261/Asp135 salt bridge that may be useful for allosteric drug discovery.

Regulation of alanine racemase activity by carboxylates and the d-type substrate d-alanine.

Alanine racemases (ALRs) are essential for d-alanine (d-Ala) production in bacteria, and many ALRs have a conserved carbamylated lysine residue in the active site. Although short-chain carboxylates inhibit ALRs harbouring this lysine residue as substrate analogues, in an ALR variant with an alanine residue at this position, carboxylates behave as activators; however, this activation mechanism remains unclear. Here, we performed kinetic and structural analyses of U1ALR, an ALR from Latilactobacillus sakei UONUMA harbouring a glycine residue (Gly134) in the site of the carbamylated lysine residue. U1ALR was activated by various carboxylates and also by a G134K mutation, both of which caused a significant decrease in Km , indicating an increase in substrate affinity. The U1ALR crystal structure revealed the presence of an acetate molecule bound in a position and at an orientation resembling the conformation of the carbamylated lysine side chain observed in the structures of other ALRs. These results suggest a regulatory mechanism for U1ALR activity involving two carboxylate-binding sites: one with high affinity near Gly134, where an acetate molecule is observed in the crystal structure and carboxylate binding results in enzyme activation; the other is the substrate-binding site, where carboxylate binding inhibits enzyme activity. Furthermore, we observed no carboxylate/G134K-mediated activation in the presence of d-Ala at high concentrations, implying that d-Ala also exhibits low-affinity binding in the first carboxylate-binding site and prevents carboxylate/G134K-induced activation. Such regulation of enzyme activity by carboxylates and d-Ala may be ubiquitous in many ALRs from lactic acid bacteria sharing the same sequence characteristics.

The Short-chain Fatty Acid Propionic Acid Activates the Rcs Stress Response System Partially through Inhibition of d-Alanine Racemase.

The Enterobacterial Rcs stress response system reacts to envelope stresses through a complex two-component phosphorelay system to regulate a variety of environmental response genes, such as capsular polysaccharide and flagella biosynthesis genes. However, beyond Escherichia coli, the stresses that activate Rcs are not well-understood. In this study, we used a Rcs system-dependent luminescent transcriptional reporter to screen a library of over 240 antimicrobial compounds for those that activated the Rcs system in Serratia marcescens, a Yersiniaceae family bacterium. Using an isogenic rcsB mutant to establish specificity, both new and expected activators were identified, including the short-chain fatty acid propionic acid, which is found at millimolar levels in the human gut. Propionic acid did not reduce the bacterial intracellular pH, as was hypothesized for its antibacterial mechanism. Instead, data suggest that the Rcs-activation by propionic acid is due, in part, to an inactivation of alanine racemase. This enzyme is responsible for the biosynthesis of d-alanine, which is an amino-acid that is required for the generation of bacterial cell walls. Consistent with what was observed in S. marcescens, in E. coli, alanine racemase mutants demonstrated elevated expression of the Rcs-reporter in a d-alanine-dependent and RcsB-dependent manner. These results suggest that host gut short-chain fatty acids can influence bacterial behavior via the activation of the Rcs stress response system. IMPORTANCE The Rcs bacterial stress response system responds to envelope stresses by globally altering gene expression to profoundly impact host-pathogen interactions, virulence, and antibiotic tolerance. In this study, a luminescent Rcs-reporter plasmid was used to screen a library of compounds for activators of Rcs. Among the strongest inducers was the short-chain fatty acid propionic acid, which is found at high concentrations in the human gut. This study suggests that gut short-chain fatty acids can affect both bacterial virulence and antibiotic tolerance via the induction of the Rcs system.

Evolution and properties of alanine racemase from Synechocystis sp. PCC6803.

Alanine racemase (EC 5.1.1.1) depends on pyridoxal 5'-phosphate and catalyses the interconversion between L- and D-Ala. The enzyme is responsible for the biosynthesis of D-Ala, which is an essential component of the peptidoglycan layer of bacterial cell walls. Phylogenetic analysis of alanine racemases demonstrated that the cyanobacterial enzyme diverged before the separation of gram-positive and gram-negative enzymes. This result is interesting considering that the peptidoglycans observed in cyanobacteria seem to combine the properties of those in both gram-negative and gram-positive bacteria. We cloned the putative alanine racemase gene (slr0823) of Synechocystis sp. PCC6803 in Escherichia coli cells, expressed and purified the enzyme protein and studied its enzymological properties. The enzymatic properties of the Synechocystis enzyme were similar to those of other gram-positive and gram-negative bacterial enzymes. Alignment of the amino acid sequences of alanine racemase enzymes revealed that the conserved tyrosine residue in the active centre of most of the gram-positive and gram-negative bacterial enzymes has been replaced with tryptophan in most of the cyanobacterial enzymes. We carried out the site-directed mutagenesis involving the corresponding residue of Synechocystis enzyme (W385) and revealed that the residue is involved in the substrate recognition by the enzyme.

Three-dimensional biofilm colony growth supports a mutualism involving matrix and nutrient sharing.

Life in a three-dimensional biofilm is typical for many bacteria, yet little is known about how strains interact in this context. Here, we created essential gene CRISPR interference knockdown libraries in biofilm-forming Bacillus subtilis and measured competitive fitness during colony co-culture with wild type. Partial knockdown of some translation-related genes reduced growth rates and led to out-competition. Media composition led some knockdowns to compete differentially as biofilm versus non-biofilm colonies. Cells depleted for the alanine racemase AlrA died in monoculture but survived in a biofilm colony co-culture via nutrient sharing. Rescue was enhanced in biofilm colony co-culture with a matrix-deficient parent due to a mutualism involving nutrient and matrix sharing. We identified several examples of mutualism involving matrix sharing that occurred in three-dimensional biofilm colonies but not when cultured in two dimensions. Thus, growth in a three-dimensional colony can promote genetic diversity through sharing of secreted factors and may drive evolution of mutualistic behavior.

D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition.

The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5'-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.

Knockout of alanine racemase gene attenuates the pathogenicity of Aeromonas hydrophila.

BACKGROUND: Aeromonas hydrophila is an opportunistic pathogen of poikilothermic and homoeothermic animals, including humans. In the present study, we described the role of Alanine racemase (alr-2) in the virulence of A. hydrophila using an alr-2 knockout mutant (A.H.Δalr). RESULTS: In mouse and common carp models, the survival of animals challenged with A.H.Δalr was significantly increased compared with the wild-type (WT), and the mutant was also impaired in its ability to replicate in the organs and blood of infected mice and fish. The A.H.Δalr significantly increased phagocytosis by macrophages of the mice and fish. These attenuation effects of alr-2 could be complemented by the addition of D-alanine to the A.H.Δalr strain. The histopathology results indicated that the extent of tissue injury in the WT-infected animals was more severe than in the A.H.Δalr-infected groups. The expression of 9 virulence genes was significantly down-regulated, and 3 outer membrane genes were significantly up-regulated in A.H.Δalr. CONCLUSIONS: Our data suggest that alr-2 is essential for the virulence of A. hydrophila. Our findings suggested alanine racemase could be applied in the development of new antibiotics against A. hydrophila.

Biochemical characterization and mutational analysis of alanine racemase from Clostridium perfringens.

Clostridium perfringens is a gram-positive, anaerobic, pathogenic bacterium that can cause a wide range of diseases in humans, poultry and agriculturally important livestock. A pyridoxal-5-phosphate-dependent alanine racemase with a function in the racemization of d- and l-alanine is an attractive drug target for C. perfringens and other pathogens due to its absence in animals and humans. In this study alanine racemase from C. perfringens (CPAlr) was successfully expressed and purified in Escherichia coli and biochemically characterized. The purified CPAlr protein was a dimeric PLP-dependent enzyme with high substrate specificity. The optimal racemization temperature and pH were 40°C and 8.0, respectively. The kinetic parameters Km and kcat of CPAlr, determined by HPLC at 40°C were 19.1 mM and 17.2 s-1 for l-alanine, and 10.5 mM and 8.7 s-1 for d-alanine, respectively. Gel filtration chromatographic analysis showed that the molecular weight of mutant Y359A was close to monomeric form, suggesting that the inner layer residue Tyr359 might play an essential role in dimer-formation. Furthermore, the mutation at residues Asp171 and Tyr359 resulted in a dramatic increase in Km value and/or decreased in kcat value, indicating that the middle and inner layer residues Asp171 and Tyr359 of CPAlr might have the key role in substrate binding, catalytic activity or oligomerization state through the hydrogen-bonding interaction with the pentagonal ring waters and/or PLP cofactor.

Overexpression of a newly identified d-amino acid transaminase in Mycobacterium smegmatis complements glutamate racemase deletion.

Glutamate racemase (MurI) has been proposed as a target for anti-tuberculosis drug development based on the inability of ΔmurI mutants of Mycobacterium smegmatis to grow in the absence of d-glutamate. In this communication, we identify ΔmurI suppressor mutants that are detected during prolonged incubation. Whole genome sequencing of these ΔmurI suppressor mutants identified the presence of a SNP, located in the promoter region of MSMEG_5795. RT-qPCR and transcriptional fusion analyses revealed that the ΔmurI suppressor mutant overexpressed MSMEG_5795 14-fold compared to the isogenic wild-type. MSMEG_5795, which is annotated as 4-amino-4-deoxychorismate lyase (ADCL) but which also has homology to d-amino acid transaminase (d-AAT), was expressed, purified and found to have d-AAT activity and to be capable of producing d-glutamate from d-alanine. Consistent with its d-amino acid transaminase function, overexpressed MSMEG_5795 is able to complement both ΔmurI deletion mutants and alanine racemase (Δalr) deletion mutants, thus confirming a multifunctional role for this enzyme in M. smegmatis.

The role of the CroR response regulator in resistance of Enterococcus faecalis to D-cycloserine is defined using an inducible receiver domain.

Enterococcus faecalis is an opportunistic multidrug-resistant human pathogen causing severe nosocomial infections. Previous investigations revealed that the CroRS two-component regulatory pathway likely displays a pleiotropic role in E. faecalis, involved in virulence, macrophage survival, oxidative stress response as well as antibiotic resistance. Therefore, CroRS represents an attractive potential new target for antibiotherapy. In this report, we further explored CroRS cellular functions by characterizing the CroR regulon: the 'domain swapping' method was applied and a CroR chimera protein was generated by fusing the receiver domain from NisR to the output domain from CroR. After demonstrating that the chimera CroR complements a croR gene deletion in E. faecalis (stress response, virulence), we conducted a global gene expression analysis using RNA-Seq and identified 50 potential CroR targets involved in multiple cellular functions such as cell envelope homeostasis, substrate transport, cell metabolism, gene expression regulation, stress response, virulence and antibiotic resistance. For validation, CroR direct binding to several candidate targets was demonstrated by EMSA. Further, this work identified alr, the gene encoding the alanine racemase enzyme involved in E. faecalis resistance to D-cycloserine, a promising antimicrobial drug to treat enterococcal infections, as a member of the CroR regulon.

Role of Alanine Racemase Mutations in Mycobacterium tuberculosis d-Cycloserine Resistance.

A screening of more than 1,500 drug-resistant strains of Mycobacterium tuberculosis revealed evolutionary patterns characteristic of positive selection for three alanine racemase (Alr) mutations. We investigated these mutations using molecular modeling, in vitro MIC testing, as well as direct measurements of enzymatic activity, which demonstrated that these mutations likely confer resistance to d-cycloserine.

Identification and elucidation of in vivo function of two alanine racemases from Pseudomonas putida KT2440.

The genome of Pseudomonas putida KT2440 contains two open reading frames (ORFs), PP_3722 and PP_5269, that encode proteins with a Pyridoxal phosphate binding motif and a high similarity to alanine racemases. Alanine racemases play a key role in the biosynthesis of D-alanine, a crucial amino acid in the peptidoglycan layer. For these ORFs, we generated single and double mutants and found that inactivation of PP_5269 resulted in D-alanine auxotrophy, while inactivation of PP_3722 did not. Furthermore, as expected, the PP_3722/PP_5269 double mutant was a strict auxotroph for D-alanine. These results indicate that PP_5269 is an alr allele and that it is the essential alanine racemase in P. putida. We observed that the PP_5269 mutant grew very slowly, while the double PP_5269/PP_3722 mutant did not grow at all. This suggests that PP_3722 may replace PP_5269 in vivo. In fact, when the ORF encoding PP_3772 was cloned into a wide host range expression vector, ORF PP_3722 successfully complemented P. putida PP_5269 mutants. We purified both proteins to homogeneity and while they exhibit similar KM values, the Vmax of PP_5269 is fourfold higher than that of PP_3722. Here, we propose that PP_5269 and PP_3722 encode functional alanine racemases and that these genes be named alr-1 and alr-2 respectively.

High-level production of α-amylase by manipulating the expression of alanine racamase in Bacillus licheniformis.

OBJECTIVES: To improve target protein production by manipulating expression levels of alanine racemase in Bacillus licheniformis. RESULTS: The gene of dal was identified to be responsible for alanine racemase function. Based on the selection marker of dal, a food-grade expression system was constructed in B. licheniformis, and effects of different dal expression levels mediated by promoters on α-amylase production were investigated. The highest α-amylase activity (155 U/ml) was obtained in BL10D/pP43SAT-PtetDal, increased by 27% compared with that of the control strain BL10/pP43SAT in tetracycline-based system (123 U/ml). Moreover, the dal transcriptional level was not correlated positively with that of amyL. CONCLUSIONS: A food-grade system for high-level production of α-amylase was constructed in B. licheniformis, revealing that expression levels of selection marker significantly affected target protein production.

A Clostridium difficile alanine racemase affects spore germination and accommodates serine as a substrate.

Clostridium difficile has become one of the most common bacterial pathogens in hospital-acquired infections in the United States. Although C. difficile is strictly anaerobic, it survives in aerobic environments and transmits between hosts via spores. C. difficile spore germination is triggered in response to certain bile acids and glycine. Although glycine is the most effective co-germinant, other amino acids can substitute with varying efficiencies. Of these, l-alanine is an effective co-germinant and is also a germinant for most bacterial spores. Many endospore-forming bacteria embed alanine racemases into their spore coats, and these enzymes are thought to convert the l-alanine germinant into d-alanine, a spore germination inhibitor. Although the C. difficile Alr2 racemase is the sixth most highly expressed gene during C. difficile spore formation, a previous study reported that Alr2 has little to no role in germination of C. difficile spores in rich medium. Here, we hypothesized that Alr2 could affect C. difficile l-alanine-induced spore germination in a defined medium. We found that alr2 mutant spores more readily germinate in response to l-alanine as a co-germinant. Surprisingly, d-alanine also functioned as a co-germinant. Moreover, we found that Alr2 could interconvert l- and d-serine and that Alr2 bound to l- and d-serine with ∼2-fold weaker affinity to that of l- and d-alanine. Finally, we demonstrate that l- and d-serine are also co-germinants for C. difficile spores. These results suggest that C. difficile spores can respond to a diverse set of amino acid co-germinants and reveal that Alr2 can accommodate serine as a substrate.

The alr-groEL1 operon in Mycobacterium tuberculosis: an interplay of multiple regulatory elements.

Threonylcarbamoyladenosine is a universally conserved essential modification of tRNA that ensures translational fidelity in cellular milieu. TsaD, TsaB and TsaE are identified as tRNA-A37-threonylcarbamoyl (t6A)-transferase enzymes that have been reconstituted in vitro, in few bacteria recently. However, transcriptional organization and regulation of these genes are not known in any of these organisms. This study describes the intricate architecture of a complex multicistronic alr-groEL1 operon, harboring essential genes, namely tsaD, tsaB, tsaE, groES, groEL1, and alr (required for cell wall synthesis), and rimI encoding an N-α- acetyltransferase in Mycobacterium tuberculosis. Using northern blotting, RT-PCR and in vivo fluorescence assays, genes alr to groEL1 were found to constitute an ~6.3 kb heptacistronic operon with multiple internal promoters and an I-shaped intrinsic hairpin-like cis-regulatory element. A strong promoter PtsaD within the coding sequence of rimI gene is identified in M. tuberculosis, in addition. The study further proposes an amendment in the known bicistronic groESL1 operon annotation by providing evidence that groESL1 is co-transcribed as sub-operon of alr-groEL1 operon. The architecture of alr-groEL1 operon, conservation of the genetic context and a mosaic transcriptional profile displayed under various stress conditions convincingly suggest the involvement of this operon in stress adaptation in M. tuberculosis.

Structural and functional characterization of the alanine racemase from Streptomyces coelicolor A3(2).

The conversion of l-alanine (L-Ala) into d-alanine (D-Ala) in bacteria is performed by pyridoxal phosphate-dependent enzymes called alanine racemases. D-Ala is an essential component of the bacterial peptidoglycan and hence required for survival. The Gram-positive bacterium Streptomyces coelicolor has at least one alanine racemase encoded by alr. Here, we describe an alr deletion mutant of S. coelicolor which depends on D-Ala for growth and shows increased sensitivity to the antibiotic d-cycloserine (DCS). The crystal structure of the alanine racemase (Alr) was solved with and without the inhibitors DCS or propionate, at 1.64 Å and 1.51 Å resolution, respectively. The crystal structures revealed that Alr is a homodimer with residues from both monomers contributing to the active site. The dimeric state of the enzyme in solution was confirmed by gel filtration chromatography, with and without L-Ala or d-cycloserine. The activity of the enzyme was 66 ± 3 U mg-1 for the racemization of L- to D-Ala, and 104 ± 7 U mg-1 for the opposite direction. Comparison of Alr from S. coelicolor with orthologous enzymes from other bacteria, including the closely related d-cycloserine-resistant Alr from S. lavendulae, strongly suggests that structural features such as the hinge angle or the surface area between the monomers do not contribute to d-cycloserine resistance, and the molecular basis for resistance therefore remains elusive.

Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics.

Histidine is a unique amino acid with an imidazole side chain in which both of the nitrogen atoms are capable of serving as a proton donor and proton acceptor in hydrogen bonding interactions. In order to probe the functional role of histidine involved in hydrogen bonding networks, fine-tuning the hydrogen bonding potential of the imidazole side chain is required but not feasible through traditional mutagenesis methods. Here, we show that two close mimetics of histidine, 3-methyl-histidine and thiazole alanine, can be genetically encoded using engineered pyrrolysine incorporation machinery. Replacement of the three histidine residues predicted to be involved in an extended charge-relay system in alanine racemase with 3-methyl-histidine or thiazole alanine shows a dramatic loss in the enzyme's catalytic efficiency, implying the role of this extended charge-relay system in activating the active site residue Y265, a general acid/base catalyst in the enzyme.

Mechanistic and Evolutionary Insights from the Reciprocal Promiscuity of Two Pyridoxal Phosphate-dependent Enzymes.

Enzymes that utilize the cofactor pyridoxal 5'-phosphate play essential roles in amino acid metabolism in all organisms. The cofactor is used by proteins that adopt at least five different folds, which raises questions about the evolutionary processes that might explain the observed distribution of functions among folds. In this study, we show that a representative of fold type III, the Escherichia coli alanine racemase (ALR), is a promiscuous cystathionine ß-lyase (CBL). Furthermore, E. coli CBL (fold type I) is a promiscuous alanine racemase. A single round of error-prone PCR and selection yielded variant ALR(Y274F), which catalyzes cystathionine ß-elimination with a near-native Michaelis constant (Km = 3.3 mm) but a poor turnover number (kcat ≈10 h(-1)). In contrast, directed evolution also yielded CBL(P113S), which catalyzes l-alanine racemization with a poor Km (58 mm) but a high kcat (22 s(-1)). The structures of both variants were solved in the presence and absence of the l-alanine analogue, (R)-1-aminoethylphosphonic acid. As expected, the ALR active site was enlarged by the Y274F substitution, allowing better access for cystathionine. More surprisingly, the favorable kinetic parameters of CBL(P113S) appear to result from optimizing the pKa of Tyr-111, which acts as the catalytic acid during l-alanine racemization. Our data emphasize the short mutational routes between the functions of pyridoxal 5'-phosphate-dependent enzymes, regardless of whether or not they share the same fold. Thus, they confound the prevailing model of enzyme evolution, which predicts that overlapping patterns of promiscuity result from sharing a common multifunctional ancestor.

Secretion of d-alanine by Escherichia coli.

Escherichia coli has an l-alanine export system that protects the cells from toxic accumulation of intracellular l-alanine in the presence of l-alanyl-l-alanine (l-Ala-l-Ala). When a DadA-deficient strain was incubated with 6.0 mM l-Ala-l-Ala, we detected l-alanine and d-alanine using high-performance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with d-amino acid oxidase resulted in the disappearance of a signal corresponding to d-alanine. Additionally, the culture supernatant enabled a d-alanine auxotroph to grow without d-alanine supplementation, confirming that the signal detected by HPLC was authentic d-alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, alr or dadX, the extracellular level of d-alanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow from l-alanine to d-alanine enhanced d-alanine secretion. When high-density DadA-deficient cells preloaded with l-Ala-l-Ala were treated with 20 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), secretion of both l-alanine and d-alanine was enhanced ~twofold compared with that in cells without CCCP treatment. In contrast, the ATPase inhibitor dicyclohexylcarbodiimide did not exert such an effect on the l-alanine and d-alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the l-alanine exporter AlaE accumulated [3H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [3H]D-alanine was strongly inhibited by CCCP. These results indicate that E. coli has a transport system(s) that exports d-alanine and that this function is most likely modulated by proton electrochemical potential.