Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

A lysine racemase (lyr) gene was isolated from a soil metagenome by functional complementation for the first time by using Escherichia coli BCRC 51734 cells as the host and d-lysine as the selection agent. The lyr gene consisted of a 1,182-bp nucleotide sequence encoding a protein of 393 amino acids with a molecular mass of about 42.7 kDa. The enzyme exhibited higher specific activity toward lysine in the l-lysine-to-d-lysine direction than in the reverse reaction.Amino acids are the building blocks of proteins and play an important role in the regulation of the metabolism of living organisms. Among two enantiomers of naturally occurring amino acids, l-amino acids are predominant in living organisms, while d-amino acids are found in both free and bound states in various organisms like bacteria (36), yeasts (35), plants (47), insects (11), mammals (17), bivalves (39), and fish (28). The d-amino acids are mostly endogenous and produced by racemization from their counterparts by the action of a racemase. Thus, the amino acid racemases are involved in d-amino acid metabolism (29, 46). Since the discovery of alanine racemase in 1951 (42), several racemases toward amino acids, such as those for glutamate, threonine, serine, aspartate, methionine, proline, arginine, and phenylalanine, have been reported in bacteria, archaea, and eukaryotes, including mammals (1, 2, 15, 30, 31, 44). They are classified into two groups: pyridoxal 5′-phosphate (PLP)-dependent and PLP-independent enzymes (9, 36).Lysine racemase (Lyr, EC 5.1.1.5) was first reported in Proteus vulgaris ATCC 4669 (19) and proposed to be involved in the lysine degradation of bacterial cells (5, 19). Catabolism of lysine occurs via two parallel pathways. In one of the pathways, δ-aminovalerate is the key metabolite, whereas in the other l-lysine is racemized to d-lysine, and l-pipecolate and α-aminoadipate (AMA) are the key metabolites (5). d-Lysine catabolism proceeds through a series of cyclized intermediates which are necessary to regenerate an α-amino acid and comprise the following metabolites (AMA pathway): d-lysine→α-keto-ɛ-amino caproate→Δ¹-piperideine-2-carboxylate→pipecolate→Δ¹-piperideine-6-carboxylate→α-amino-δ-formylcaproate→α-AMA→α-ketoadipate (6, 7, 12, 27). The final product is converted to α-ketoglutarate via a series of coenzyme A derivatives and subsequently participates as an intermediate in the Krebs cycle. This pathway suggests that the biological function of d-lysine in the bacteria is that of a carbon or nitrogen source. Racemization of added l-lysine to d-lysine by whole cells of Proteus spp. and Escherichia spp. (19) and by the cell extract of Pseudomonas putida ATCC 15070 (5, 20) has been found. However, the enzyme has not been purified to homogeneity, and thus, its molecular and catalytic characteristics, including its gene structure, have not been elucidated. In this study, we explored a metagenomic library constructed from a garden soil to isolate a novel Lyr enzyme. After expression in Escherichia coli, the purified enzyme was characterized in terms of optimal pH and temperature, thermal stability, and racemization activity.     

The Aminolysis Reaction of Streptomyces S9 Aminopeptidase Promotes the Synthesis of Diverse Prolyl Dipeptides

Prolyl dipeptide synthesis by S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) has been demonstrated. In the synthesis, S9AP-St preferentially used l-Pro-OBzl as the acyl donor, yielding synthesized dipeptides having an l-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acyl acceptor. Furthermore, S9AP-St produced cyclo (l-Pro-l-His) with a conversion ratio of substrate to cyclo (l-Pro-l-His) higher than 40%.Some proline-containing dipeptides and their cyclic analogs exhibit biological activity. For example, cyclo (l-arginyl-d-proline) [c(lR-dP)] is known to act as a specific inhibitor of family 18 chitinase (4, 10). A cyclic peptide, c(lP-lH), produced by the cleavage of thyrotropin-releasing hormone protects against oxidative stress, promotes cytoprotection (6, 7), and exhibits antihyperglycemic activity (11).Some serine peptidases exhibit peptide bond formation (i.e., aminolysis of esters, thioesters, and amides) in accordance with their hydrolytic activity (2, 14). The exchange of catalytic Ser for Cys to engineer the serine endopeptidase into “transpeptidase” for peptide bond formation has been well characterized (3, 5). Our recent approach confirmed the wide distribution of family S9 aminopeptidases that have catalytic Ser in actinomycetes (12). Of them, we obtained S9 aminopeptidase from Streptomyces thermocyaneoviolaceus NBRC14271 (S9AP-St). The enzyme was engineered into “transaminopeptidase” by exchange of catalytic Ser for Cys, and its aminolytic activity was evaluated (13). The engineered enzyme, designated as aminolysin-S, can synthesize hydrophobic dipeptides through an aminolysis reaction. However, aminolysin-S was unable to synthesize peptides containing proline. Although the report of aminolysin-S demonstrated that S9AP-St shows no aminolysis reaction toward limited substrates, details of its characteristics remain unknown. This study verified the peptide synthetic activity of S9AP-St, demonstrating that S9AP-St can synthesize widely varied prolyl dipeptides through an aminolysis reaction. The report also shows that S9AP-St is applicable to the synthesis of a biologically active peptide—c(lP-lH).     

Substrate Specificity of a Mannose-6-Phosphate Isomerase from Bacillus subtilis and Its Application in the Production of l-Ribose

The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co²⁺. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter⁻¹ h⁻¹.l-Ribose is a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, and it is not abundant in nature (5, 19). l-Ribose has been produced mainly by chemical synthesis from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, or d-mannono-1,4-lactone (2, 17, 23). Biological l-ribose manufacture has been investigated using ribitol or l-ribulose. Recently, l-ribose was produced from ribitol by a recombinant Escherichia coli containing an NAD-dependent mannitol-1-dehydrogenase (MDH) with a 55% conversion yield when 100 g/liter ribitol was used in a 72-h fermentation (18). However, the volumetric productivity of l-ribose in the fermentation is 28-fold lower than that of the chemical method synthesized from l-arabinose (8). l-Ribulose has been biochemically converted from l-ribose using an l-ribose isomerase from an Acinetobacter sp. (9), an l-arabinose isomerase mutant from Escherichia coli (4), a d-xylose isomerase mutant from Actinoplanes missouriensis (14), and a d-lyxose isomerase from Cohnella laeviribosi (3), indicating that l-ribose can be produced from l-ribulose by these enzymes. However, the enzymatic production of l-ribulose is slow, and the enzymatic production of l-ribose from l-ribulose has been not reported.Sugar phosphate isomerases, such as ribose-5-phosphate isomerase, glucose-6-phosphate isomerase, and galactose-6-phosphate isomerase, work as general aldose-ketose isomerases and are useful tools for producing rare sugars, because they convert the substrate sugar phosphates and the substrate sugars without phosphate to have a similar configuration (11, 12, 21, 22). l-Ribose isomerase from an Acinetobacter sp. (9) and d-lyxose isomerase from C. laeviribosi (3) had activity with l-ribose, d-lyxose, and d-mannose. Thus, we can apply mannose-6-phosphate (EC 5.3.1.8) isomerase to the production of l-ribose, because there are no sugar phosphate isomerases relating to l-ribose and d-lyxose. The production of the expensive sugar l-ribose (bulk price, $1,000/kg) from the rare sugar l-ribulose by mannose-6-phosphate isomerase may prove to be a valuable industrial process, because we have produced l-ribulose from the cheap sugar l-arabinose (bulk price, $50/kg) using the l-arabinose isomerase from Geobacillus thermodenitrificans (20) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic representation for the production of l-ribulose from l-arabinose by G. thermodenitrificans l-arabinose isomerase and the production of l-ribose from l-ribulose by B. subtilis mannose-6-phosphate isomerase.In this study, the gene encoding mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in E. coli. The substrate specificity of the recombinant enzyme for various aldoses and ketoses was investigated, and l-ribulose exhibited the highest activity among all pentoses and hexoses. Therefore, mannose-6-phosphate isomerase was applied to the production of l-ribose from l-ribulose.     

Methanococci Use the Diaminopimelate Aminotransferase (DapL) Pathway for Lysine Biosynthesis

The pathway of lysine biosynthesis in the methanococci has not been identified previously. A variant of the diaminopimelic acid (DAP) pathway uses diaminopimelate aminotransferase (DapL) to catalyze the direct conversion of tetrahydrodipicolinate (THDPA) to ll-DAP. Recently, the enzyme DapL (MTH52) was identified in Methanothermobacter thermautotrophicus and shown to belong to the DapL1 group. Although the Methanococcus maripaludis genome lacks a gene that can be unambiguously assigned a DapL function based on sequence similarity, the open reading frame MMP1527 product shares 30% amino acid sequence identity with MTH52. A Δmmp1527 deletion mutant was constructed and found to be a lysine auxotroph, suggesting that this DapL homolog in methanococci is required for lysine biosynthesis. In cell extracts of the M. maripaludis wild-type strain, the specific activity of DapL using ll-DAP and α-ketoglutarate as substrates was 24.3 ± 2.0 nmol min⁻¹ mg of protein⁻¹. The gene encoding the DapL homolog in Methanocaldococcus jannaschii (MJ1391) was cloned and expressed in Escherichia coli, and the protein was purified. The maximum activity of MJ1391 was observed at 70°C and pH 8.0 to 9.0. The apparent K_ms of MJ1391 for ll-DAP and α-ketoglutarate were 82.8 ± 10 μM and 0.42 ± 0.02 mM, respectively. MJ1391 was not able to use succinyl-DAP or acetyl-DAP as a substrate. Phylogenetic analyses suggested that two lateral gene transfers occurred in the DapL genes, one from the archaea to the bacteria in the DapL2 group and one from the bacteria to the archaea in the DapL1 group. These results demonstrated that the DapL pathway is present in marine methanogens belonging to the Methanococcales.Two lysine biosynthesis pathways evolved separately in organisms, the diaminopimelic acid (DAP) and aminoadipic acid (AAA) pathways. The DAP pathway synthesizes l-lysine from aspartate and pyruvate, and diaminopimelic acid is an intermediate. This pathway is utilized by most bacteria, some archaea, some fungi, some algae, and plants (28, 29). The AAA pathway synthesizes l-lysine from α-ketoglutarate and acetyl coenzyme A (acetyl-CoA), and α-aminoadipic acid is an intermediate. This pathway is utilized by most fungi, some algae, the bacterium Thermus thermophilus, and probably some archaea, such as Sulfolobus, Thermoproteus, and Pyrococcus (27, 36). No organism is known to possess both pathways.There are four known variations of the DAP pathway in bacteria: the succinylase, acetylase, aminotransferase, and dehydrogenase pathways (Fig. ​(Fig.1).1). These pathways share the steps converting l-aspartate to l-2,3,4,5-tetrahydrodipicolinate (THDPA), but the subsequent steps leading to the production of meso-diaminopimelate, the immediate precursor of l-lysine, are different. The succinylase pathway acylates THDPA with succinyl-CoA to generate N-succinyl-ll-2-amino-6-ketopimelate and forms meso-DAP by subsequent transamination, desuccinylation, and epimerization. This pathway is utilized by proteobacteria and many firmicutes and actinobacteria (12, 14, 20, 29). The acetylase pathway is analogous to the succinylase pathway but uses N-acetyl intermediates. This pathway is limited to certain Bacillus species, in which the corresponding genes have not been identified (33, 39). The aminotransferase pathway converts THDPA directly to ll-DAP by diaminopimelate aminotransferase (DapL) without acylation. This pathway is shared by cyanobacteria (19), chlamydia (24), the archaeon Methanothermobacter thermautotrophicus (15, 18), and the plant Arabidopsis thaliana (19). The dehydrogenase pathway forms meso-DAP directly from THDPA, NADPH, and NH₄⁺ by using diaminopimelate dehydrogenase (Ddh). This pathway is utilized by some Bacillus and Brevibacterium species and Corynebacterium glutamicum (25, 26, 40). Most bacteria use only one of the four variants, although certain bacteria, such as C. glutamicum and Bacillus macerans, possess both the succinylase and dehydrogenase pathways (3, 30).Open in a separate windowFIG. 1.Variations in the DAP pathway for lysine biosynthesis. 1, succinylase pathway; 2, acetylase pathway; 3, aminotransferase pathway; 4, dehydrogenase pathway. Abbreviations and designations: THDPA, l-2,3,4,5-tetrahydrodipicolinate; l,l-DAP, ll-2,6-diaminopimelate; meso-DAP, meso-2,6-diaminopimelate; LysC, aspartate kinase; Asd, aspartate semialdehyde dehydrogenase; DapA, dihydrodipicolinate synthase; DapB, dihydrodipicolinate reductase; DapD, THDPA succinylase; DapC, succinyl-DAP aminotransferase; DapE, succinyl-DAP desuccinylase; DapF, DAP epimerase; LysA, DAP decarboxylase; DapL, ll-DAP aminotransferase; Ddh, DAP dehydrogenase.The diaminopimelate aminotransferase (DapL) catalyzes the transfer of an amino group from l-glutamate to THDPA, forming ll-DAP (19, 24). It uses pyridoxal 5′-phosphate (PLP) as a coenzyme and has constrained substrate specificity. DapL is not closely related to the DapC/ArgD aminotransferase, which functions in the succinylase pathway. Comparative genomic analysis identified dapL homologs in both bacterial and archaeal genomes. Homologs of dapD and dapE have not been found in genomes with dapL homologs, suggesting that transamination of THDPA does not require succinylation in these organisms (18). Phylogenetic analysis also suggested classification of DapL into two groups, DapL1 and DapL2, which share ∼30% amino acid sequence identity (18). These two groups both exhibit DapL activity, and they cannot be differentiated by kinetic properties (18, 37). The distribution of the two groups is not obviously associated with specific prokaryotic lineages (18).Methanogens are strictly anaerobic archaea that obtain all or most of their energy for growth from the production of large quantities of methane. All methanogens belong to the Euryarchaeota and are currently classified in six orders: Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyrales, and Methanocellales (23, 41, 42). Biochemical studies of Methanocaldococcus jannaschii and Methanococcus voltae belonging to Methanococcales, Methanospirillum hungatei belonging to Methanomicrobiales, and Methanothermobacter thermautotrophicus belonging to Methanobacteriales suggested that these organisms derive their l-lysine from a DAP pathway, but the studies did not discriminate among the four DAP pathway variations (2, 9, 10, 32). Genome sequence analysis also suggested a DAP pathway in Methanosarcina mazei belonging to Methanosarcinales (8). Recent studies identified a dapL homolog belonging to the DapL1 group in M. thermautotrophicus. The gene product complemented an Escherichia coli dapD dapE double mutant and catalyzed the transamination of DAP to THDPA, suggesting that Methanobacteriales use the DapL pathway for l-lysine biosynthesis (15, 18). Homologs of asd, dapA, dapB, dapF, and lysA have been identified in the genomes of M. maripaludis and M. jannaschii belonging to the Methanococcales, but homologs responsible for the conversion of THDPA to ll-DAP have not been annotated (4, 17). Here we identified methanococcal DapL homologs and demonstrated that the DapL pathway is present in Methanococcales.     

Zif,the Zoocin A Immunity Factor,Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a d-alanyl-l-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three l-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.During streptococcal peptidoglycan synthesis, monomer subunits are generated inside the cell, with nonribosomal peptidyl transferases responsible for the addition of amino acids onto the epsilon amino group of lysine in the subunits. These nonribosomal peptidyl transferases are part of the FemABX family of proteins, some of which have been implicated in penicillin resistance (5, 26). In Streptococcus pneumoniae peptidoglycan synthesis, MurM attaches either an l-alanine or an l-serine to the epsilon amino group of lysine, and MurN then adds an l-alanine (11, 26).Zoocin A is a d-alanyl-l-alanine endopeptidase produced by Streptococcus equi subsp. zooepidemicus 4881 that hydrolyzes peptidoglycan cross bridges of susceptible streptococci (12). Zoocin A has two functional domains (18). The N-terminal catalytic domain (CAT) has high degrees of similarity to several other bacteriolytic endopeptidases, including the staphylolytic enzyme lysostaphin. The C-terminal target recognition domain (TRD), which facilitates binding of the enzyme to peptidoglycan (1), has very little similarity to any characterized conserved domain.Producer cell immunity to zoocin A is due to zif (zoocin A immunity factor), which is adjacent to zooA on the chromosome and is transcribed divergently (4). Zif has high degrees of similarity to MurM and MurN and also to the lysostaphin resistance protein and other FemABX-like immunity proteins (23). Previously characterized FemABX-like immunity proteins provide resistance to peptidoglycan cross-bridge hydrolases by inserting an amino acid different from those specified by the normal FemABX-like proteins (6, 9, 15, 25), whereas Zif does not (4). It has been shown previously that Zif-specified resistance to zoocin A is an intrinsic characteristic of the peptidoglycan layer (12). Therefore, Zif must modify the peptidoglycan layer in a novel way that provides resistance to zoocin A. In the present study, Zif was shown to insert an additional l-alanine into the peptidoglycan cross bridges, which inhibited both binding of the zoocin A TRD and the ability of the zoocin A CAT to hydrolyze the cross bridge.     

Functional and Biochemical Analysis of the Chlamydia trachomatis Ligase MurE

Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-l-Ala-d-Glu:meso-diaminopimelic acid (UDP-MurNAc-l-Ala-d-Glu:meso-A₂pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that the murE gene from Chlamydia trachomatis is capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-l-Ala-d-Glu:meso-A₂pm ligase activity. Recombinant MurE from C. trachomatis (MurE_Ct) was overproduced in and purified from E. coli in order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-l-Ala-d-Glu as the nucleotide substrate, MurE_Ct demonstrated ATP-dependent meso-A₂pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, the ll and dd isomers of A₂pm, d-lysine) were also recognized by MurE_Ct. However, the activities for these amino acid substrates were weaker than that for meso-A₂pm. The specificity of MurE_Ct for three possible C. trachomatis peptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relative k_cat/K_m ratios for UDP-MurNAc-l-Ala-d-Glu, UDP-MurNAc-l-Ser-d-Glu, and UDP-MurNAc-Gly-d-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid is meso-A₂pm. However, due to the lack of specificity of MurE_Ct for nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.Chlamydiae cause serious respiratory tract and genital infections in humans (9). They are obligately intracellular gram-negative bacteria, with a unique biphasic development cycle. Elementary bodies (EBs) are the infectious form of the organism and invade susceptible host cells. Once internalized, EBs differentiate into reticulate bodies (RBs), which have the capacity to divide (39, 40). The RBs are fragile and pleomorphic, whereas EBs are comparatively rigid and stable (19, 39). After repeated cycles of binary fission, the RBs differentiate into EBs, and the host cell lyses, releasing infectious EBs (1).In contrast to the vast majority of eubacteria, chlamydiae lack detectable amounts of peptidoglycan (PG), an essential polymer. PG is a giant macromolecule composed of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues cross-linked by short peptides. It determines the shape of bacteria, protects the cell from lysis due to internal osmotic pressure, and also plays a role in cell division. However, PG has not been detected in EBs (14, 20) or RBs (5).Although chlamydiae appear to lack PG, they contain penicillin-binding proteins and are sensitive to antibiotics that inhibit PG synthesis (5, 40). The Chlamydia trachomatis genome contains most of the genes coding for proteins involved in, or associated with, PG synthesis (54). Chlamydial MurA, MurC-Ddl, and the MurC domain of the latter fusion protein are active in vitro and complement Escherichia coli mutants deficient in the respective enzymes (23, 32, 33). Furthermore, proteomic analysis reveals that the murE gene product, which was assigned as UDP-MurNAc-l-Ala-d-Glu:meso-diaminopimelic acid ligase (UDP-MurNAc-l-Ala-d-Glu:meso-A₂pm ligase), is expressed in RBs (52).MurE ligases catalyze the addition of the third amino acid residue to the peptide chain of PG. This residue, generally a diamino acid, is usually meso-A₂pm for gram-negative bacteria and bacilli, and l-lysine for gram-positive bacteria, although other amino acids (for example, l-ornithine, meso-lanthionine, ll-A₂pm, l-diaminobutyric acid, or l-homoserine) occur in certain species (6, 50, 57). In many organisms, the third residue of the peptide chain participates in PG cross-linking; consequently, the MurE enzyme is highly specific for the relevant amino acid so as to avoid incorporation of incorrect amino acids into the macromolecule, which could result in deleterious morphological changes and cell lysis (35). Crystallization of MurE from E. coli (MurE_Ec) has permitted analysis of the structural basis for this high specificity (22). Sequence alignments of different MurE orthologues have also revealed the specific consensus sequences DNPR and D(D/N)P(N/A) located in the binding pockets for meso-A₂pm and l-Lys, respectively (11, 17). Chlamydia trachomatis MurE (MurE_Ct) possesses the DNPR motif, which suggests that it adds meso-A₂pm (17). However, there are no experimental data to confirm this prediction.In this paper we report for the first time the overproduction and purification of MurE_Ct, as well as a detailed investigation of its in vivo and in vitro biochemical properties. These studies contribute to our understanding of the nature and properties of the PG biosynthetic enzymes in chlamydiae and do indeed suggest that MurE_Ct has meso-A₂pm ligase activity.     

Characterization of the Dicarboxylate Transporter DctA in Corynebacterium glutamicum

Transporters of the dicarboxylate amino acid-cation symporter family often mediate uptake of C₄-dicarboxylates, such as succinate or l-malate, in bacteria. A member of this family, dicarboxylate transporter A (DctA) from Corynebacterium glutamicum, was characterized to catalyze uptake of the C₄-dicarboxylates succinate, fumarate, and l-malate, which was inhibited by oxaloacetate, 2-oxoglutarate, and glyoxylate. DctA activity was not affected by sodium availability but was dependent on the electrochemical proton potential. Efficient growth of C. glutamicum in minimal medium with succinate, fumarate, or l-malate as the sole carbon source required high dctA expression levels due either to a promoter-up mutation identified in a spontaneous mutant or to ectopic overexpression. Mutant analysis indicated that DctA and DccT, a C₄-dicarboxylate divalent anion/sodium symporter-type transporter, are the only transporters for succinate, fumarate, and l-malate in C. glutamicum.In bacteria, the uptake of dicarboxylates, such as the tricarboxylic acid (TCA) cycle intermediates succinate, fumarate, and l-malate, is mediated by transporters of different protein families. Whereas Dcu-type transporters facilitate dicarboxylate uptake under anaerobic conditions, the most common aerobic dicarboxylate transporters are members of the dicarboxylate amino acid-cation symporter (DAACS), divalent anion sodium symporter (DASS), tripartite ATP-independent periplasmic (TRAP), and CitMHS transporter families. DAACS transporters are responsible for C₄-dicarboxylate uptake under aerobic conditions in various bacteria, e.g., DctA from Escherichia coli, Bacillus subtilis, or Rhizobium leguminosarum, and are involved in different physiological functions (2, 4, 27, 41). The first described member of the TRAP family is the C₄-dicarboxylate transporter DctPQM from Rhodobacter capsulatus, which facilitates substrate uptake by the use of an extracytoplasmic solute receptor (8). An example of the DASS family, members of which occur in bacteria, as well in eukaryotes, is the well-characterized transporter SdcS from Staphylococcus aureus (13). Members of the CitHMS family import citrate in symport with the cation Mg²⁺ or Ca²⁺. Whereas E. coli possesses one DctA and four different Dcu carriers, no Dcu transporter-encoding genes were found in Corynebacterium glutamicum (16, 19), which is used for the industrial production of amino acids, such as glutamate (33) or l-lysine (39), and is capable of succinate and l-lactate production under oxygen deprivation conditions. A dctA gene was annotated (19); however, C. glutamicum is not able to utilize succinate, malate, or fumarate as a sole carbon source. The uptake systems CitH and TctCBA have been characterized recently as citrate uptake systems (3, 26). Interestingly, we and others have shown that C. glutamicum possesses a DASS family transporter (DccT) for uptake of the C₄-dicarboxylates succinate, fumarate, and l-malate (36, 40). Spontaneous mutants showing fast growth in succinate or fumarate minimal medium were isolated and shown to possess promoter-up mutations in the dccT gene (40). In l-malate minimal medium, these spontaneous mutants showed relatively slow growth, and the affinity of DccT for succinate and fumarate was found to be 5- and 12-fold higher than for l-malate, respectively (40). These findings prompted us to search for other uptake systems for l-malate in C. glutamicum. Here, we describe the identification and characterization of the DAACS family protein DctA from C. glutamicum as a proton motive force-driven uptake system for C₄-dicarboxylate intermediates of the TCA cycle. Additionally, we compare both uptake systems, DccT and DctA, from C. glutamicum.     

Requirements for Germination of Clostridium sordellii Spores In Vitro

Clostridium sordellii is a spore-forming, obligately anaerobic, Gram-positive bacterium that can cause toxic shock syndrome after gynecological procedures. Although the incidence of C. sordellii infection is low, it is fatal in most cases. Since spore germination is believed to be the first step in the establishment of Bacilli and Clostridia infections, we analyzed the requirements for C. sordellii spore germination in vitro. Our data showed that C. sordellii spores require three structurally different amino acids and bicarbonate for maximum germination. Unlike the case for Bacilli species, d-alanine had no effect on C. sordellii spore germination. C. sordellii spores germinated only in a narrow pH range between 5.7 and 6.5. In contrast, C. sordellii spore germination was significantly less sensitive to temperature changes than that of the Bacilli. The analysis of the kinetics of C. sordellii spore germination showed strong allosteric behavior in the binding of l-phenylalanine and l-alanine but not in that of bicarbonate or l-arginine. By comparing germinant apparent binding affinities to their known in vivo concentrations, we postulated a mechanism for differential C. sordellii spore activation in the female reproductive tract.Clostridium sordellii is an anaerobic, Gram-positive, spore-forming bacterium that is commonly found in soil and in the intestines of animals (4). Many C. sordellii strains are nonpathogenic; however, virulent strains cause lethal infections in several animal species, such as hemorrhagic enteritis in foals, sheep, and cattle (5, 10, 16, 28), omphalitis in foals (43), and wound infection in humans (4, 35).C. sordellii also can cause life-threatening necrotizing infections after gynecological procedures (4). In addition, fatal cases of C. sordellii endometritis following medical abortion with a mifepristone-misoprostol combination have been reported recently (13, 19, 56). The increased use of mifepristone-misoprostol for medical abortion may result in larger numbers of C. sordellii infections (38, 40).Although C. sordellii rarely has been identified in the genital tract, a correlation between gynecological procedures and C. sordellii-mediated toxic shock syndrome is apparent (19). Pregnancy, childbirth, or abortion may predispose some women to acquire C. sordellii in the vaginal tract (19). Under these conditions, C. sordellii infections result in an almost 100% mortality rate.Since there is no national system for tracking and reporting complications associated with gynecological procedures, the identification of the true rates of reproductive tract infections in women is not readily available (8). Therefore, the number of known C. sordellii-associated infections, although low, may be underreported (19, 29). Furthermore, unsafe abortion practices in developing countries cause large mortality rates due to complicating infections (24, 34). In many cases, however, the causative agent of the abortion-associated sepsis have not been characterized (24). Thus, the worldwide morbidity and mortality associated with C. sordellii infections is not currently known.C. sordellii produces several virulence factors. The two major toxins are the lethal toxin (TcsL) and the hemorrhagic toxin (37, 46). The lethal toxin produced by C. sordellii is causally involved in enteritis of domestic animals and in systemic toxicity following infections of humans (46). Furthermore, TcsL is associated with rapid mortality in C. sordellii endometritis rodent models (26). Interestingly, TcsL cytopathic effects are increased at low pH, a characteristic found in the vaginal tract (48). The hemorrhagic toxin is not well characterized, but it has been reported to cause dermal and intestinal necrosis in guinea pigs (6, 52).C. sordellii, like other Bacilli and Clostridia species, has the ability to form metabolically dormant spores that are extremely resistant to environmental stresses, such as heat, radiation, and toxic chemicals (42, 55). Upon encountering a suitable environment, spores germinate into vegetative cells, the form that is responsible for toxin production and disease onset (39, 54).In most cases, the germination process initially is triggered by the detection of low-molecular-weight germinants by a sensitive biosensor (39, 54). This sensor consists of a proteinaceous germination (Ger) receptor encoded, in general, by a tricistronic operon. Spore germination requirements have been studied most extensively for Bacilli and can be initiated by a variety of factors, including amino acids, sugars, and nucleosides (20, 30).Spore germination in the Clostridia generally requires combinations of multiple germinants. The germination of spores of proteolytic Clostridium botulinum types A and B was triggered by a defined three-component mixture comprised of l-alanine (or l-cysteine), l-lactate (or sodium thioglycolate), and sodium bicarbonate (3). In contrast, the optimum germination of spores of nonproteolytic C. botulinum types B, E, and F required binary combinations of l-alanine-l-lactate, l-cysteine-l-lactate, and l-serine-l-lactate (45).Clostridium difficile is a human pathogen that can cause fulminant colitis (11). Interestingly, C. difficile does not encode any known Ger receptors (53). However, it is likely that germination receptors exist, because C. difficile spores must germinate in order to complete their life cycle. While C. difficile germination receptors remain elusive, the spores of C. difficile germinate in rich medium supplemented with bile salts (62). More recently, taurocholate (a bile salt) and glycine (an amino acid) were shown to act as cogerminants for C. difficile spore germination (57, 61).Clostridium bifermentans is a close relative of C. sordellii (14). The minimum requirement for C. bifermentans spore germination was the presence of l-alanine, l-phenylalanine, and l-lactate (59). In addition, an unknown factor present in yeast extract was suggested to enhance germination (59). However, the Ger receptors involved in C. bifermentans spore germination are not known.Even though many Bacilli and Clostridia species use similar metabolites as germinants, the mechanisms of germinant recognition remain to be elucidated. Unfortunately, the multimeric interactions of Ger receptor complexes and the hydrophobic nature of the Ger receptor subunits have hindered our understanding of the mechanism of germinant recognition.To understand the molecular determinants of germinant recognition, we recently applied kinetic methods to study bacterial spore germination (1, 2, 18). Spore germination can be analyzed quantitatively by fitting optical density (OD) decreases to the Michaelis-Menten equation (2). The kinetic parameters obtained allow the determination of the apparent binding affinity (K_m) of spores for the different cogerminants and the maximum rate of spore germination (V_max). In these instances, K_m refers to the concentration of substrate required to reach half of the maximal germination rate. These parameters can, in turn, be used to determine the mechanism of germination and potential interactions between germination receptors. Furthermore, by comparing apparent K_m values to germinant concentrations in vivo, models for spore-germinant complex distribution can be proposed, and rate-limiting steps for the germination process can be derived. Thus, kinetic analysis can yield information on spore activation even if the identities of the germination receptors are not known.Using this procedure, we were able to determine the mechanism for Bacillus anthracis germination with inosine and l-alanine. In turn, this information was used to design nucleoside analogs that inhibit B. anthracis spore germination in vitro and protect macrophages from anthrax cytotoxicity (2).Since C. sordellii germination receptors have not been identified, we used chemical probes and kinetic methods to investigate the conditions necessary for spore germination. We found that C. sordellii spores germinate better at slightly acidic pH. Furthermore, germination rates varied slightly from 25 to 40°C. We also found that C. sordellii spores have an absolute requirement for a small amino acid, a basic amino acid, an aromatic amino acid, and bicarbonate (NaHCO₃) for efficient germination. Kinetic analysis showed allosteric interaction for the putative l-phenylalanine and l-alanine germination receptors. In contrast, l-arginine or bicarbonate recognition followed typical Michaelis-Menten kinetics. The implication of germinant recognition and host environment is discussed.     

Efficient Production of Optically Pure d-Lactic Acid from Raw Corn Starch by Using a Genetically Modified l-Lactate Dehydrogenase Gene-Deficient and α-Amylase-Secreting Lactobacillus plantarum Strain

In order to achieve direct and efficient fermentation of optically pure d-lactic acid from raw corn starch, we constructed l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 α-amylase (AmyA). The resulting strain produced only d-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct d-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct d-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct d-lactic acid fermentation from raw starch.Poly-lactic acid (PLA) is an important agro-based plastic that can be produced from inexpensive, renewable, and abundantly available biomass resources, including starchy materials. These resources have advantages over limited oil- and fossil-based sources, as they do not result in any net carbon dioxide release to the atmosphere (7). Recently, stereocomplex PLA, which is composed of both poly-l- and -d-lactic acid, has been attracting much attention due to its high thermostability. Stereocomplex-type polymers show a melting point (ca. 230°C) that is approximately 50°C higher than that of the respective single polymers (8). Therefore, d-lactic acid, in addition to l-lactic acid, which has been the focus of production to date, is of significant importance.Lactic acid bacteria (LAB) are promising microorganisms for the efficient production of lactic acid from various sugars, such as glucose, sucrose, and lactose. However, when starchy materials are used as a carbon source, they must be saccharified by physicochemical and enzymatic treatment because most LAB cannot utilize starchy materials directly (13). This makes the whole process less economically viable. Therefore, many researchers have examined the direct production of lactic acid from starchy materials by using wild amylolytic LAB (ALAB) (6, 24, 25) or genetically modified amylase-producing LAB (15, 16). Although d-lactic acid has been produced by fermentation from pretreated substrates such as rice starch (5) and by simultaneous saccharification and fermentation from cellulose (23), there have been no reports on the direct production of d-lactic acid from starchy materials. This is due to a lack of d-lactic acid-producing ALAB and difficulties in gene manipulation of d-lactic acid-producing LAB, such as Lactobacillus delbrueckii (22).We focused on Lactobacillus plantarum, which is an industrially important strain due to its environmental flexibility and its ability to assimilate a wide range of carbohydrates (9). In recent years, several gene manipulation methods for Lactobacillus plantarum have been established (18, 19). Moreover, the complete genome sequence has been decoded for L. plantarum NCIMB 8826 (9). Based on whole-genome analysis, L. plantarum possesses two types of lactate dehydrogenase (LDH), l-LDH and d-LDH, which convert pyruvate into l- and d-lactic acid, respectively. Ferain et al. (4) reported that chromosomal deletion in the ldhL1 gene of L. plantarum NCIMB 8826 provoked an absence of l-LDH activity and produced d-lactic acid from glucose.In the present study, to produce d-lactic acid directly from starch, we constructed an l-LDH-deficient, α-amylase-secreting L. plantarum strain. The engineered strain expressed α-amylase from Streptococcus bovis 148 (AmyA) (20) and efficiently degraded raw starch with the aid of a C-terminal starch-binding domain (11). Using this strain, we achieved the direct and efficient fermentation of optically pure d-lactic acid from raw corn starch.     

Cadaverine Covalently Linked to Peptidoglycan Is Required for Interaction between the Peptidoglycan and the Periplasm-Exposed S-Layer-Homologous Domain of Major Outer Membrane Protein Mep45 in Selenomonas ruminantium

The peptidoglycan of Selenomonas ruminantium is covalently bound to cadaverine (PG-cadaverine), which likely plays a significant role in maintaining the integrity of the cell surface structure. The outer membrane of this bacterium contains a 45-kDa major protein (Mep45) that is a putative peptidoglycan-associated protein. In this report, we determined the nucleotide sequence of the mep45 gene and investigated the relationship between PG-cadaverine, Mep45, and the cell surface structure. Amino acid sequence analysis showed that Mep45 is comprised of an N-terminal S-layer-homologous (SLH) domain followed by α-helical coiled-coil region and a C-terminal β-strand-rich region. The N-terminal SLH domain was found to be protruding into the periplasmic space and was responsible for binding to peptidoglycan. It was determined that Mep45 binds to the peptidoglycan in a manner dependent on the presence of PG-cadaverine. Electron microscopy revealed that defective PG-cadaverine decreased the structural interactions between peptidoglycan and the outer membrane, consistent with the proposed role for PG-cadaverine. The C-terminal β-strand-rich region of Mep45 was predicted to be a membrane-bound unit of the 14-stranded β-barrel structure. Here we propose that PG-cadaverine possesses functional importance to facilitate the structural linkage between peptidoglycan and the outer membrane via specific interaction with the SLH domain of Mep45.Polyamines, the ubiquitous polycationic compounds composed of a hydrocarbon backbone with multiple amino groups, exist in all living cells and participate in a wide variety of biological reactions, including DNA, RNA, and protein synthesis (34). However, it has been revealed that some strictly anaerobic eubacteria belonging to the Veillonellaceae family, such as Selenomonas ruminantium, Veillonella alcalescens, Veillonella parvula, and Anaerovibrio lipolyticus, possess polyamines covalently linked to their peptidoglycan (PG) as an essential constituent (8, 16, 17). S. ruminantium possesses a peptidoglycan associated with cadaverine. Cadaverine binds covalently to the α-carboxyl group of the d-glutamic acid residue of peptidoglycan by one of its two amino groups, and the other amino group remains as a free cation (15). In this bacterium, cadaverine is synthesized constitutively from lysine by lysine/ornithine decarboxylase (LDC/ODC [EC 4.1.1.18]), a bifunctional enzyme that decarboxylates both l-lysine and l-ornithine at similar K_m and V_max values (35, 36) and is transferred to a d-glutamic acid residue by a particulate enzyme designated as lipid intermediate:diamine transferase (20). The cadaverine synthesis by LDC/ODC is completely inhibited by dl-α-difluoromethyllysine (DFML) or dl-α-difluoromethylornithine (DFMO), which inhibits the decarboxylating activity toward both l-lysine and l-ornithine (35), and the prevention of the cadaverine synthesis in S. ruminantium was shown to lead to the significant decrease of the amount of the cadaverine covalently linked to peptidoglycan (PG-cadaverine) and result in the growth inhibition (17). Since this inhibitory effect accompanies a drastic morphological change of the cells resulting in an aberrant cell surface structure, PG-cadaverine has been assumed to play a significant role in maintaining the integrity of the cell surface (17).The cell surface structure of S. ruminantium has a typical Gram-negative three-layer organization, comprising a cytoplasmic membrane, peptidoglycan layer, and outer membrane (18). However, it contains neither the free nor bound form of murein-lipoprotein (19), which plays an important role in the structural linkage between the outer membrane and peptidoglycan, thereby maintaining the structural integrity of the cell surface structures of Gram-negative bacteria (5, 33). The Escherichia coli lpo mutant that lacks murein-lipoprotein becomes hypersensitive to EDTA, resulting in rapid cell lysis upon exposure to EDTA. In contrast, S. ruminantium shows no cell lysis, even in the presence of high concentrations of EDTA, despite the absence of murein-lipoprotein (19). One possible interpretation for these findings was the assumption that PG-cadaverine associates with the structural connection between the outer membrane and peptidoglycan, thereby replacing the function of murein-lipoprotein with an outer membrane component or components. Nevertheless, the factors in the outer membrane interacting with PG-cadaverine have not been identified.The outer membrane of S. ruminantium contains a 45-kDa major protein (Mep45), which has been proposed to be a peptidoglycan-associating protein (18, 19). Kalmokoff et al. reported that the major outer membrane protein of S. ruminantium OB268, which is similar to Mep45 in size, contains an N-terminal surface-layer homology (SLH) domain (13), a putative functional domain that interacts with cell wall components (27). These findings prompted us to investigate the Mep45 major outer membrane protein of S. ruminantium as a putative outer membrane component interacting with PG-cadaverine. In this report, we characterize the Mep45 protein and its interactions with PG-cadaverine and prove their involvement in the structural linkage between the outer membrane and peptidoglycan.     

Adaptive Evolution of Escherichia coli K-12 MG1655 during Growth on a Nonnative Carbon Source,l-1,2-Propanediol

Laboratory adaptive evolution studies can provide key information to address a wide range of issues in evolutionary biology. Such studies have been limited thus far by the inability of workers to readily detect mutations in evolved microbial strains on a genome scale. This limitation has now been overcome by recently developed genome sequencing technology that allows workers to identify all accumulated mutations that appear during laboratory adaptive evolution. In this study, we evolved Escherichia coli K-12 MG1655 with a nonnative carbon source, l-1,2-propanediol (l-1,2-PDO), for ∼700 generations. We found that (i) experimental evolution of E. coli for ∼700 generations in 1,2-PDO-supplemented minimal medium resulted in acquisition of the ability to use l-1,2-PDO as a sole carbon and energy source so that the organism changed from an organism that did not grow at all initially to an organism that had a growth rate of 0.35 h⁻¹; (ii) six mutations detected by whole-genome resequencing accumulated in the evolved E. coli mutant over the course of adaptive evolution on l-1,2-PDO; (iii) five of the six mutations were within coding regions, and IS5 was inserted between two fuc regulons; (iv) two major mutations (mutations in fucO and its promoter) involved in l-1,2-PDO catabolism appeared early during adaptive evolution; and (v) multiple defined knock-in mutant strains with all of the mutations had growth rates essentially matching that of the evolved strain. These results provide insight into the genetic basis underlying microbial evolution for growth on a nonnative substrate.Evolution of microorganisms in the laboratory offers the possibility of relating acquired mutations to increased fitness of the organism under the conditions used. Complete identification of mutations over defined evolutionary periods is necessary to fully understand the evolutionary change because spontaneous mutation is the foundational biological source of phenotypic variation (52). Since microbes grow rapidly and have large population sizes and since ancestors can be preserved by freezing them for later direct comparison of evolved types, laboratory evolution using microorganisms provides a powerful context for studying the genetics of evolutionary adaptation (5, 12, 14, 19, 43) due to the advent of new technologies for genome-wide detection of mutations (30, 33). A large number of studies of experimental evolution with various microbes have been carried out using natural carbon sources, especially glucose (12, 19, 47, 55), since glucose is the preferred carbon and energy source for most bacteria and eukaryotic cells (4, 50). Recently, a few studies have investigated the adaptive evolution of Escherichia coli at the genetic and metabolic levels with gluconeogenic carbon sources, including lactate (34) and glycerol (20). Compared to experimental evolution with native carbon sources, microorganisms might be more capable of adapting to various nonnative carbon compounds because microorganisms are able to adapt to environmental changes by using a number of strategies to meet their growth requirements and to achieve optimal overall performance in the new conditions (20, 21, 34). However, a comprehensive analysis of the genetic basis of adaptation to nonnative carbon sources has not been performed.The K-12 MG1655 strain of E. coli is not able to utilize l-1,2-propanediol (l-1,2-PDO) as a sole carbon and energy source. However, E. coli has an enzyme, l-1,2-PDO oxidoreductase (POR), which is involved in fermentative l-fucose metabolism and catalyzes the oxidation of l-1,2-PDO to l-lactaldehyde (Fig. ​(Fig.11 A). The E. coli POR is encoded by the fucO gene of the fucose regulon (11, 23), which consists of two divergent operons (fucAO and fucPIKUR) under positive control of FucR (Fig. ​(Fig.1B)1B) (9). FucR is activated by fuculose-1-phosphate, which is the inducer of the fuc regulon (3). In E. coli, fucose metabolism is initiated by the sequential actions of a permease (encoded by fucP), an isomerase (encoded by fucI), a kinase (encoded by fucK), and an aldolase (encoded by fucA). The aldolase catalyzes the cleavage of fuculose-1-phosphate to dihydroxyacetone phosphate and l-lactaldehyde. Under aerobic respiratory conditions, l-lactaldehyde is oxidized to l-lactate by an NAD-linked aldehyde dehydrogenase with broad functions (encoded by aldA). l-Lactate is then oxidized to pyruvate by a flavin adenine dinucleotide (FAD)-dependent l-lactate dehydrogenase (encoded by the lldD gene of the lldPRD operon [formerly the lctPRD operon]). Under anaerobic fermentative conditions, however, redox balance requires sacrifice of the l-lactaldehyde as a hydrogen acceptor at the expense of NADH (Fig. ​(Fig.1A).1A). This reaction is catalyzed by the POR. The terminal fermentation product, l-1,2-PDO, is then released by a permease (57). Although the POR catalyzes the oxidation of l-1,2-PDO to l-lactaldehyde, l-1,2-PDO cannot be utilized by wild-type (WT) E. coli as a sole carbon source under aerobic conditions because this compound cannot induce expression of the fuc regulon (11). Indeed, the fuc regulon was not expressed under any conditions when a database of 213 expression profiles produced in our laboratory was examined (38). Furthermore, even if the POR is expressed, it is oxidatively inactivated by a metal-catalyzed oxidation (MCO) mechanism (7).Open in a separate windowFIG. 1.Metabolic pathway and fuc regulon for l-fucose and l-1,2-PDO. (A) Metabolic pathway for l-fucose and l-1,2-PDO. In E. coli, fucose metabolism is initiated by the sequential actions of a permease (encoded by fucP), an isomerase (encoded by fucI), a kinase (encoded by fucK), and an aldolase (encoded by fucA). The aldolase catalyzes cleavage of fuculose-1-phosphate to dihydroxyacetone phosphate and l-lactaldehyde. Under aerobic respiratory conditions, the l-lactaldehyde is further oxidized by a series of enzymes to pyruvate, which subsequently enters central metabolism. Under anaerobic fermentative conditions, the l-lactaldehyde is reduced to l-1,2-PDO by oxidoreductase (encoded by fucO). (B) Genetic organization of the fuc regulon. The fuc regulon for l-fucose uptake and metabolism consists of two divergent operons, fucAO and fucPIKUR.Sridhara et al. (48) previously described E. coli mutants that were isolated from an E. coli K-12 derivative treated with the mutagen ethyl methanesulfonate and were able to grow aerobically on l-1,2-PDO as a sole carbon source. Previous studies showed that an IS5 insertion between the fucAO and fucPIKUR operons caused constitutive expression of the fucAO operon (9, 41) at a level that enabled the E. coli mutant to grow on l-1,2-PDO. In addition, mutations resulting in increased resistance to MCO under aerobic conditions were found in the N-terminal domain of POR (39). However, at present, little is known about the accumulated genome-wide mutations and their effects on the fitness in E. coli that has acquired the ability to use l-1,2-PDO because previous studies have focused on mutations in POR and its regulatory region.In an attempt to investigate the genetic basis of adaptive evolution of E. coli during growth on l-1,2-PDO, we first isolated an E. coli mutant able to use l-1,2-PDO using experimental evolution without a mutagen, and we then characterized this evolved E. coli mutant. Using whole-genome sequencing, we identified all accumulated mutations of the evolved E. coli mutant related to the known ancestor and also determined the fitness benefits and phenotypic behaviors of the mutations discovered. Our results offer a systematic view of the genetic basis underlying microbial adaptation to a nonnative substrate.     

Design of a Potent d-Peptide HIV-1 Entry Inhibitor with a Strong Barrier to Resistance

The HIV gp41 N-trimer pocket region is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Here, we report on the design of a pocket-specific d-peptide, PIE12-trimer, that is extraordinarily elusive to resistance and characterize its inhibitory and structural properties. d-Peptides (peptides composed of d-amino acids) are promising therapeutic agents due to their insensitivity to protease degradation. PIE12-trimer was designed using structure-guided mirror-image phage display and linker optimization and is the first d-peptide HIV entry inhibitor with the breadth and potency required for clinical use. PIE12-trimer has an ultrahigh affinity for the gp41 pocket, providing it with a reserve of binding energy (resistance capacitor) that yields a dramatically improved resistance profile compared to those of other fusion inhibitors. These results demonstrate that the gp41 pocket is an ideal drug target and establish PIE12-trimer as a leading anti-HIV antiviral candidate.The HIV envelope protein (Env) mediates viral entry into cells (11). Env is cleaved into surface (gp120) and transmembrane (gp41) subunits that remain noncovalently associated to form trimeric spikes on the virion surface (16). gp120 recognizes target cells by interacting with cellular receptors, while gp41 mediates membrane fusion. Peptides derived from heptad repeats near the N and C termini of the gp41 ectodomain (N and C peptides) interact in solution to form a six-helix bundle, representing the postfusion structure (3, 55, 56). In this structure, N peptides form a central trimeric coiled coil (N trimer), creating grooves into which C peptides bind. This structure, in conjunction with the dominant-negative inhibitory properties of exogenous N and C peptides, suggests a mechanism for Env-mediated entry (10, 22, 58-60).During entry, gp41 forms an extended prehairpin intermediate that leaves the exposed N-trimer region vulnerable to inhibition for several minutes (18, 35). This intermediate ultimately collapses as the C-peptide regions bind to the N-trimer grooves to form a trimer of hairpins (six-helix bundle), juxtaposing viral and cellular membranes and inducing fusion. Enfuvirtide (Fuzeon), the only clinically approved HIV fusion inhibitor, is a C peptide that binds to part of the N-trimer groove and prevents six-helix bundle formation in a dominant-negative manner (61). Enfuvirtide is active in patients with multidrug resistance to other classes of inhibitors and is a life-prolonging option for these patients (30, 31). However, enfuvirtide use is restricted to salvage therapy due to several limitations, including (i) high dosing requirements (90 mg, twice-daily injections), (ii) high cost (∼$30,000/year/patient in the United States), and (iii) the rapid emergence of resistant strains (21, 47).A deep hydrophobic pocket at the base of the N-trimer groove is an especially attractive inhibitory target because of its high degree of conservation (3, 12, 48), poor tolerance to substitution (4, 34), and critical role in membrane fusion (2). Indeed, this region is conserved at both the amino acid level (for gp41 function in membrane fusion) and the nucleotide level (for the structured RNA region of the Rev-responsive element). Enfuvirtide binds to the N-trimer groove just N terminal to the pocket and is significantly more susceptible to resistance mutations than 2nd-generation C-peptide inhibitors, such as T-1249, that also bind to the pocket (8, 13, 29, 44, 46, 47, 58).Peptide design, molecular modeling, and small-molecule screening have produced a diverse set of compounds that interact with the gp41 pocket and inhibit HIV-1 entry with modest potency, but often with significant cytotoxicity (7, 14, 15, 17, 23, 24, 26, 34, 51, 54). The first direct evidence that pocket-specific binders are sufficient to inhibit HIV entry came with the discovery of protease-resistant d-peptides identified using mirror-image phage display (12). In this technique, a phage library is screened against a mirror-image version of the target protein (synthesized using d-amino acids) (50). By symmetry, mirror images (d-peptides) of the discovered sequences will bind to the natural l-peptide target. As the mirror images of naturally occurring l-peptides, d-peptides cannot be digested by natural proteases. Protease resistance provides d-peptides theoretical treatment advantages of extended survival in the body and possible oral bioavailability (41, 42, 49).These 1st-generation d-peptide entry inhibitors possess potency against a laboratory-adapted isolate (HXB2) at low to mid-μM concentrations (12). We previously reported an affinity-matured 2nd-generation d-peptide called PIE7, pocket-specific inhibitor of entry 7 (57). A trimeric version of PIE7 is the first high-affinity pocket-specific HIV-1 inhibitor and has potency against X4-tropic (HXB2) and R5-tropic (BaL) strains at sub-nM concentrations. However, significant further optimization is required to create a robust clinical candidate for two reasons. First, this d-peptide is much less potent (requiring high nM concentrations) against JRFL, a primary R5-tropic strain. Therefore, improved PIE potency is necessary to combat diverse primary strains. Second, by improving the affinity of our inhibitors for the pocket target, we hope to provide a reserve of binding energy that will delay the emergence of drug resistance, as described below.We and others have reported a potency plateau for some gp41-based fusion inhibitors that is likely imposed by the transient exposure of the prehairpin intermediate (9, 27, 53, 57). For very high-affinity inhibitors, association kinetics (rather than affinity) limits potency so that two inhibitors with significantly different affinities for the prehairpin intermediate can have similar antiviral potencies. We proposed that overengineering our d-peptides with substantial affinity beyond this potency plateau would provide a reserve of binding energy that would combat affinity-disrupting resistance mutations (57). Such a resistance capacitor should also prevent the stepwise accumulation of subtle resistance mutations in Env by eliminating the selective advantage that such mutants would otherwise confer.Here, we report on the design and characterization of a 3rd-generation pocket-specific d-peptide, PIE12-trimer, with ∼100,000-fold improved target binding compared to that of the best previous d-peptide, significantly broadened inhibitory potency, and an enhanced resistance capacitor that provides a strong barrier to viral resistance. We achieved this increased potency via structure-guided phage display and crosslinker optimization. PIE12-trimer has a dramatically improved resistance profile compared to the profiles of earlier d-peptides, as well as those of enfuvirtide and T-1249. These results validate the resistance capacitor hypothesis and establish PIE12-trimer as a leading anti-HIV therapeutic candidate.     

Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.l-Arabinose isomerase (l-AI) is an enzyme that mediates in vivo isomerization between l-arabinose and l-ribulose as well as in vitro isomerization of d-galactose and d-tagatose (20). l-Ribulose (l-erythro-pentulose) is a rare and expensive ketopentose sugar (1) that can be used as a precursor for the production of other rare sugars of high market value, such as l-ribose. Despite being a common metabolic intermediate in different organisms, l-ribulose is scarce in nature. The market for rare and unnatural sugars has been growing, especially in the sweetener and pharmaceutical industries. For example, several modified nucleosides derived from l-sugars have been shown to act as potent antiviral agents and are also useful in antigen therapy. Derivatives of rare sugars have also been used as agents against hepatitis B virus and human immunodeficiency virus (2, 22).For these reasons, interest in the enzymology of rare sugars has also been increasing. Various forms of l-AI from a variety of organisms have been characterized, and some have shown potential for industrial use. Several highly thermotolerant enzyme forms from Thermotoga maritima (12), Thermotoga neapolitana (10), Bacillus stearothermophilus (18), Thermoanaerobacter mathranii (9), and Lactobacillus plantarum (5) have been characterized previously. All of these reported l-AIs tend to have broad specificity, although a few l-AIs with high degrees of substrate specificity for l-arabinose have also been documented.The enzyme properties of l-AIs have been examined by engineering several forms by error-prone PCR and site-directed mutagenesis. Galactose conversion was reportedly enhanced 20% following site-directed introduction of a double mutation (C450S-N475K) into l-AI (16). Error-prone PCR manipulation of l-AI from Geobacillus stearothermophilus resulted in a shift in temperature specificity from 60 to 65°C and increased isomerization activity (11). All of these previously reported mutational studies have been aimed at improving enzymatic properties for industrial application. However, even though the three-dimensional (3D) structure of Escherichia coli l-AI has been determined previously (15), few new structural studies have been performed to decipher the reaction mechanism of this enzyme. Rhimi et al. (19) have reported an important role for D308, F329, E351, and H446 in catalysis, as indicated by findings from site-directed mutagenesis. Nonetheless, detailed analysis of the important molecular determinants controlling the catalytic activities of the l-AIs is still lacking.Previously, we have reported the cloning and characterization of a novel l-AI from Bacillus licheniformis (17). This enzyme can be distinguished from other l-AIs by its wide pH range, high degree of substrate specificity for l-arabinose, and extremely high turnover rate. In the present paper, we report the identification of an important amino acid residue responsible for the catalytic efficiency of l-AIs, as determined by a systematic screening process composed of sequence alignment and molecular dynamics (MD) simulation, followed by site-directed mutagenesis. Using the crystal structure of E. coli l-AI as a template, we have built a 3D model of B. licheniformis l-AI. Analysis of the 3D model of B. licheniformis l-AI docked with l-arabinose, followed by a systematic screening process, showed that Y333 interacted with the substrate, suggesting that this residue in B. licheniformis l-AI may be essential for catalysis. We further characterized the role of Y333 in B. licheniformis l-AI binding of and catalytic efficiency for l-arabinose.