Addition Of D-alanine Research Articles

D-amino Acids Ameliorate Experimental Colitis and Cholangitis by Inhibiting Growth of Proteobacteria: Potential Therapeutic Role in Inflammatory Bowel Disease

D-amino acids, the chiral counterparts of protein L-amino acids, were primarily produced and utilized by microbes, including those in the human gut. However, little was known about how orally administered or microbe-derived D-amino acids affected the gut microbial community or gut disease progression. The ratio of D- to L-amino acids was analyzed in feces and blood from patients with ulcerative colitis (UC) and healthy controls. Also, composition of microbe was analyzed from patients with UC. Mice were treated with D-amino acid in dextran sulfate sodium colitis model and liver cholangitis model. The ratio of D- to L-amino acids was lower in the feces of patients with UC than that of healthy controls. Supplementation of D-amino acids ameliorated UC-related experimental colitis and liver cholangitis by inhibiting growth of Proteobacteria. Addition of D-alanine, a major building block for bacterial cell wall formation, to culture medium inhibited expression of the ftsZ gene required for cell fission in the Proteobacteria Escherichia coli and Klebsiella pneumoniae, thereby inhibiting growth. Overexpression of ftsZ restored growth of E.coli even when D-alanine was present. We found that D-alanine not only inhibited invasion of pathological K.pneumoniae into the host via pore formation in intestinal epithelial cells but also inhibited growth of E.coli and generation of antibiotic-resistant strains. D-amino acids might have potential for use in novel therapeutic approaches targeting Proteobacteria-associated dysbiosis and antibiotic-resistant bacterial diseases by means of their effects on the intestinal microbiota community.

Immunogenicity of Recombinant-Deficient Lactobacillus casei with Complementary Plasmid Expressing Alanine Racemase Gene and Core Neutralizing Epitope Antigen against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED), which is caused by the porcine epidemic diarrhea virus (PEDV), has occurred worldwide and poses a serious threat to the pig industry. Intestine is the main function site of PEDV; therefore, it is important to develop an oral mucosal immunity vaccine against this virus infection. Most traditional plasmid delivery vectors use antibiotic genes as a selective marker, easily leading to antibiotic accumulation and gene contamination. In this study, to explore whether the alanine racemase gene (Alr) could be used as a screening marker and develop an efficient oral vaccine against PEDV infection, a recombinant strain was constructed using Lactobacillus casei with Alr deletion (L. casei ΔAlr W56) to deliver the Alr gene and a core-neutralizing epitope (COE) antigen. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in mice. Compared to the other strains, the recombinant bacteria were able to grow without the addition of D-alanine, revealing that Alr in the plasmid could function normally in defective bacteria. This oral mucosal vaccine would provide a useful strategy to substitute the application of antibiotics in the future and induce efficient immune responses against PEDV infection.

A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker

BackgroundFood-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized. To develop a food-grade expression system for d-psicose 3-epimerase production, we use an alanine racemase-encoding gene as selection marker in Bacillus subtilis.ResultsIn this study, the d-alanine racemase-encoding gene dal was deleted from the chromosome of B. subtilis 1A751 using Cre/lox system to generate the food-grade host. Subsequently, the plasmid-coded selection marker dal was complemented in the food-grade host, and RDPE was thus successfully expressed in dal deletion strain without addition of d-alanine. The selection appeared highly stringent, and the plasmid was stably maintained during culturing. The highest RDPE activity in medium reached 46 U/ml at 72 h which was comparable to RDPE production in kanamycin-based system. Finally, the capacity of the food-grade B. subtilis 1A751D2R was evaluated in a 7.5 l fermentor with a fed-batch fermentation.ConclusionThe alanine racemase-encoding gene can be used as a selection marker, and the food-grade expression system was suitable for heterologous proteins production in B. subtilis.

Role of the gerA operon in L-alanine germination of Bacillus licheniformisspores

BackgroundThe genome of Bacillus licheniformis DSM 13 harbours three neighbouring open reading frames showing protein sequence similarities to the proteins encoded from the Bacillus subtilis subsp. subtilis 168 gerA operon, GerAA, GerAB and GerAC. In B. subtilis, these proteins are assumed to form a germinant receptor involved in spore germination induced by the amino acid L-alanine.ResultsIn this study we show that disruption of the gerAA gene in B. licheniformis MW3 hamper L-alanine and casein hydrolysate-triggered spore germination, measured by absorbance at 600 nm and confirmed by phase contrast microscopy. This ability was restored by complementation with a plasmid-borne copy of the gerA locus. Addition of D-alanine in the casein hydrolysate germination assay abolished germination of both B. licheniformis MW3 and the complementation mutant. Germination of both B. licheniformis MW3 and the gerA disruption mutant was induced by the non-nutrient germinant Ca2+-Dipicolinic acid.ConclusionsThese results demonstrate that the B. licheniformis MW3 gerA locus is involved in germination induced by L-alanine and potentially other components present in casein hydrolysate.

The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile

The dlt operon in Gram-positive bacteria encodes proteins that are necessary for the addition of d-alanine to teichoic acids of the cell wall. The addition of d-alanine to the cell wall results in a net positive charge on the bacterial cell surface and, as a consequence, can decrease the effectiveness of antimicrobials, such as cationic antimicrobial peptides (CAMPs). Although the roles of the dlt genes have been studied for some Gram-positive organisms, the arrangement of these genes in Clostridium difficile and the life cycle of the bacterium in the host are markedly different from those of other pathogens. In the current work, we determined the contribution of the putative C. difficile dlt operon to CAMP resistance. Our data indicate that the dlt operon is necessary for full resistance of C. difficile to nisin, gallidermin, polymyxin B and vancomycin. We propose that the d-alanylation of teichoic acids provides protection against antimicrobial peptides that may be essential for growth of C. difficile in the host.

Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors

The vanR, vanS, vanH, vanA, and vanX genes of enterococcal transposon Tn1546 were introduced into the chromosome of Enterococcus faecalis JH2-2. Complementation of this portion of the van gene cluster by a plasmid encoding VanY D,D-carboxypeptidase led to a fourfold increase in the vancomycin MIC (from 16 to 64 micrograms/ml). Multicopy plasmids pAT80 (vanR vanS vanH vanA vanX) and pAT382 (vanR vanS vanH vanA vanX vanY) conferred similar levels of vancomycin resistance to JH2-2. The addition of D-alanine (100 mM) to the culture medium restored the vancomycin susceptibility of E. faecalis JH2-2/pAT80. The pentapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala partially replaced pentadepsipeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac when the strain was grown in the presence of D-alanine. In contrast, resistance mediated by pAT382 was almost unaffected by the addition of the amino acid. Expression of the vanY gene of pAT382 resulted in the formation of the tetrapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala, indicating that a portion of the cytoplasmic precursors had been hydrolyzed. These results show that VanY contributes to glycopeptide resistance in conditions in which pentapeptide is present in the cytoplasm above a threshold concentration. However, the contribution of the enzyme to high-level resistance mediated by Tn1546 appears to be moderate, probably because hydrolysis of D-alanyl-D-alanine by VanX efficiently prevents synthesis of the pentapeptide.

Analysis of the peptidoglycan of Rickettsia prowazekii.

In the present study, peptidoglycan from Rickettsia prowazekii, an obligate intracellular bacterium, was purified. The rickettsial peptidoglycan is like that of gram-negative bacteria; that is, it is sodium dodecyl sulfate insoluble, lysozyme sensitive, and composed of glutamic acid, alanine, and diaminopimelic acid in a molar ratio of 1.0:2.3:1.0. The small amount of lysine found in the peptidoglycan preparation suggests that a peptidoglycan-linked lipoprotein(s) may be present in the rickettsiae. D-Cycloserine, a D-alanine analog which inhibits the biosynthesis of bacterial cell walls, prevented rickettsial growth in mouse L929 cells at a high concentration and altered the morphology of the rickettsiae at a low concentration. These effects were prevented by the addition of D-alanine. This suggests that R. prowazekii contains D-alanine in the peptidoglycan and has D-Ala-D-Ala ligase and alanine racemase activities.

Effect of substitution of a pyridoxal phosphate-binding lysyl residue of thermostable D-amino acid aminotransferase by arginine and alanine

Lys-145 of the thermostable D-amino acid aminotransferase, which binds pyridoxal phosphate, was replaced by Ala or Arg by site-directed mutagenesis. Both mutant enzymes were purified to homogeneity; their absorption spectra indicated that both mutant enzymes contained pyridoxal phosphate bound non-covalently. Even though the standard assay method did not indicate any activity with either mutant, addition of an amino donor, D-alanine, to the Arg-145 mutant enzyme led to a slow decrease in absorption at 392 nm with a concomitant increase in absorption at 333 nm. This result suggests that the enzyme was converted into the pyridoxamine phosphate form. The amount of pyruvate formed was almost equivalent to that of the reactive pyridoxal phosphate in the mutant enzyme. Thus, the Arg-145 mutant enzyme is able to catalyze slowly the half-reaction of transamination. Exogenous amines, such as methylamine, had no effect on the half-reaction with the Arg-145 mutant enzyme. In contrast, the Ala-145 mutant enzyme neither underwent the spectral change by addition of D-alanine nor catalyzed pyruvate formation, in the absence of added amine. However, the Ala-145 mutant enzyme catalyzed the half-reaction significantly in the presence of added amine. These findings suggest that a basic amino acid residue, such as lysine or arginine, is required at position 145 for catalysis of the half-reaction. The role of the exogenous amines differs with various active-site mutant enzymes.

Controlled cell lysis and protoplast formation by enhancement of inhibitors of alanine racemase by glycine

β-D-chloroalanine (200–800 μg/ml) addition to Bacillus subtilis SB 19 caused a slight inhibition of growth in MLN broth. Inhibitor plus 1% glycine initiated rapid lysis of cells (complete in 2 hours). D-alanine (500 μg/ml) prevented lysis. D-cycloserine behaved similarly. Inhibitors in 0.5 M sucrose and 1% glycine formed stable “primed protoplasts” which were converted to the bacillary form by addition of D-alanine. Characteristics of Dal + cells with inhibitor are similar to Dal − mutants. Thus Dal + cells in the presence of alanine racemase inhibitors + glycine can be used for controlled lysis and protoplast formation (suitable for PEG transformation with plasmid DNA). Inhibitors in 1% glycine exhibit a strong antibacterial action similar to potent antibiotics which inhibit cell wall formation.

Germination of spores of Clostridium bifermentans by certain amino acids, lactate and pyruvate in the presence of sodium or potassium ions.

Spores of Clostridium bifermentans germinated in the presence of l-alanine and sodium ions. The rate of germination was not altered by the addition of d-alanine but was increased by l-α-amino-n-butyrate, l-lactate, l- or d-arginine, l-histidine, dl-ornithine, l-lysine, pyruvate, l- or d-serine, l-phenylalanine or l-proline and decreased by d-phenylalanine, l-isoleucine, glycine or l-threonine. Combinations of some organic acids such as l-phenylalanine, l-arginine, l-lactate, l-serine and glycine induced germination in the absence of l-alanine. No germination occurred in the presence of l-alanine if sodium ions were absent and replacement of sodium by potassium ions reduced the germination rate in different mixtures of organic acids. Use of different cations also altered the germination rate, although the change depended on the organic germinants present.

ALANINE: CYCLOSERINE ANTAGONISM. III. QUANTITATIVE ASPECTS AND RELATION TO HEATING OF CULTURE MEDIA.

Abstract Inhibition of growth by d-cycloserine of a clinical isolate of Escherichia coli in a synthetic alanine-free broth medium was blocked by the addition of d-alanine in equimolar or greater amounts. Heating a synthetic broth medium containing the l-isomer of alanine to 115 ° C. resulted in subversion of the antibiotic effect of d-cycloserine on E. coli. Comparable heating with d-alanine resulted in loss of capacity for antagonism toward d-cycloserine-mediated antibiosis. Chemical analysis for d-alanine showed that synthetic broth medium containing l-alanine contained d-alanine following autoclave sterilization. Three commercial broth media, in common use for conventional antibacterial agent susceptibility testing, contained d-alanine before and after autoclave sterilization.