Alr Gene Research Articles

Generation of a temperature-sensitive Edwardsiella tarda mutant and its potential as a prophylactic vaccine in olive flounder (Paralichthys olivaceus)

The aim of this study was to generate temperature-sensitive Edwardsiella tarda mutant and to evaluate potential of the mutant as a vaccine in olive flounder (Paralichthys olivaceus). A temperature-sensitive E. tarda mutant was generated by replacement of alr gene promoter with cI857-λP(R) promoter system plus another CI857 expression cassette that was driven by a constitutive promoter of E. tarda (EtPR C28-1). Growth of the mutant strain was not different to that of wild-type E. tarda under conditions of culture at 39°C. However, growth of the mutant strain was retarded by culturing at 25 or 20°C without d-alanine. To further inhibit leakage of λP(R) promoter, the mutant strain was transformed with a vector harbouring an EtPR C28-1-driven cI857 cassette (pEtPR-cI857), which resulted in more limited growth compared to the mutant without the plasmids. The level of alr gene transcription in the mutant E. tarda and the mutant harbouring pEtPR-cI857 was well coincided with the result of bacterial growth. Olive flounder fingerlings immunized with the E. tarda mutant showed decreased mortality, and a boost immunization induced 100% protection against E. tarda infection. The protection rate of fish was proportional to the serum agglutination titre against E. tarda. An attenuated E. tarda mutant induced by shifting down temperature below 30°C was firstly generated. Immunization of fish with the mutant led to protection against virulent E. tarda challenge. The results suggest that the present temperature-sensitive E. tarda mutant can be a good candidate for effective vaccines for prophylaxis of edwardsiellosis. Moreover, as the present E. tarda mutant can be cultured without supplementation of the specific nutrient and can be attenuated just by decreasing temperatures below 30°C, vaccines based on the present E. tarda mutant would be advantageous in an economical aspect.

Production of transgenic embryos through nuclear transfer using ovine fetal fibroblasts transferred with foreign genes

Human ALR gene sequence was amplified by PCR from human total DNA and inserted into pIRES(2)-EGFP vector. The bicistronic eukaryotic expression vector, pIRES-EGFP/ALR, expressing EGFP, Neo(r) and ALR genes was constructed. Sheep fetal fibroblast cells (sEFCs) were transfected with pIRES-EGFP/ALR by the induction of lipofectAMINE(TM). The positive cell clones were selected with medium containing G418 (800 μg/mL). The fluorescence of transgenic cells was examined with a confocal laser scanning microscope. The expression of ALR gene was tested by PCR, RT-PCR and immuno-histochemical staining. The transgenic cells were used as donors for nuclear transfer to enucleated ovine oocytes. Transgenic embryos were tested by confocal laser scanning microscope and immuno-histochemical staining. Results showed that the EGFP and ALR genes linked with IRES were coexpressed simultaneously in sFFCs; the blastocysts formed by nuclear transfer using tranfected donor cells are all transgenic blastocysts. EGFP, ALR and Neo(r) gene were all expressed in the transgenic embryos. In conclusion that a method to construct the positive embryos before pre-implantation which stably express ALR gene by the indication of EGFP expression has been successfully established. The application of this method can simplify the procedure of testing the targets and contribute to the efficiency increasing of transgenic domestic animal production.

Alanine racemase mutants of Mycobacterium tuberculosis require d-alanine for growth and are defective for survival in macrophages and mice

Alanine racemase (Alr) is an essential enzyme in most bacteria; however, some species (e.g. Listeria monocytogenes) can utilize d-amino acid transaminase (Dat) to generate d-alanine, which renders Alr non-essential. In addition to the conflicting reports on gene knockout of alr in Mycobacterium smegmatis, a recent study concluded that depletion of Alr does not affect the growth of M. smegmatis. In order to get an unambiguous answer on the essentiality of Alr in Mycobacterium tuberculosis and validate it as a drug target in vitro and in vivo, we have inactivated the alr gene of M. tuberculosis and found that it was not possible to generate an alr knockout in the absence of a complementing gene copy or d-alanine in the growth medium. The growth kinetics of the alr mutant revealed that M. tuberculosis requires very low amounts of d-alanine (5-10 µg ml(-1)) for optimum growth. Survival kinetics of the mutant in the absence of d-alanine indicated that depletion of this amino acid results in rapid loss of viability. The alr mutant was found to be defective for growth in macrophages. Analysis of phenotype in mice suggested that non-availability of d-alanine in mice leads to clearance of bacteria followed by stabilization of bacterial number in lungs and spleen. Additionally, reversal of d-cycloserine inhibition in the presence of d-alanine in M. tuberculosis suggested that Alr is the primary target of d-cycloserine. Thus, Alr of M. tuberculosis is a valid drug target and inhibition of Alr alone should result in loss of viability in vitro and in vivo.

A Food-Grade System for Inducible Gene Expression in Lactobacillus plantarum Using an Alanine Racemase-Encoding Selection Marker

Food-grade gene expression systems for lactic acid bacteria are useful for applications in the food industry. We describe a new food-grade host/vector system for Lactobacillus plantarum based on pSIP expression vectors and the use of the homologous alanine racemase gene (alr) as selection marker. A new series of expression vectors were constructed by exchanging the erythromycin resistance gene (erm) in pSIP vectors by the L. plantarum WCFS1 alr gene. The vectors were applied for the overexpression of β-galactosidase genes from L. reuteri L103 and L. plantarum WCFS1 in an alr deletion mutant of L. plantarum WCFS1. The expression levels obtained in this way, i.e. without the use of antibiotics, were comparable to the levels obtained with the conventional system based on selection for erythromycin resistance. The new system is suitable for the production of ingredients and additives for the food industry.

Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder ( Paralichthys olivaceus)

Two auxotrophic genes that play essential roles in bacterial cell wall biosynthesis – alanine racemase ( alr) gene and aspartate semialdehyde dehydrogenase ( asd) gene – knock-out Edwardsiella tarda ( Δalr Δasd E. tarda) was generated by the allelic exchange method to develop a combined vaccine system. Green fluorescent protein (GFP) was used as a model foreign protein, and was expressed by transformation of the mutant E. tarda with antibiotic resistant gene-free plasmids harboring cassettes for GFP and asd expression (pG02-ASD-EtPR-GFP). In vitro growth of the mutant E. tarda was similar to wild-type E. tarda when d-alanine and diaminopimelic acid (DAP) were supplemented to growth medium. However, without d-alanine and/or DAP supplementation, the mutant showed very limited growth. The Δalr Δasd E. tarda transformed with pG02-ASD-EtPR-GFP showed a similar growth pattern of wild-type E. tarda when d-alanine was supplemented in the medium, and the expression of GFP could be observed even with naked eyes. The virulence of the auxotrophic mutant E. tarda was decreased, which was demonstrated by approximately 10 6 fold increase of LD 50 dose compared to wild-type E. tarda. To assess vaccine potential of the present combined vaccine system, olive flounder ( Paralichthys olivaceus) were immunized with the GFP expressing mutant E. tarda, and analyzed protection efficacy against E. tarda challenge and antibody titers against E. tarda and GFP. Groups of fish immunized with 10 7 CFU of the Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP showed no mortality, which was irrespective to boost immunization. The cumulative mortality rates of fish immunized with 10 6 or 10 5 CFU of the mutant bacteria were lowered by a boost immunization. Fish immunized with the mutant E. tarda at doses of 10 6–10 7 CFU/fish showed significantly higher serum agglutination activities against formalin-killed E. tarda than PBS-injected control fish. Furthermore, fish immunized with 10 6–10 7 CFU/fish of the mutant E. tarda showed significantly higher ELISA titer against GFP antigen than fish in other groups. These results indicate that the present double auxotrophic genes knock-out E. tarda coupled with a heterologous antigen expression has a great strategic potential to be used as combined vaccines against various fish diseases.

Aldose Reductase Gene Polymorphisms and Diabetic Retinopathy Susceptibility

OBJECTIVEAldose reductase (ALR) is involved in diabetic microvascular damage via the polyol pathway. A recent meta-analysis found genetic variation in the ALR gene (AKR1B1) to be significantly associated with diabetic retinopathy (DR). We investigated the genetic association of AKR1B1 with DR.RESEARCH DESIGN AND METHODSThe study enrolled 909 individuals with diabetes. Participants were genotyped for an AKR1B1 (CA)n microsatellite and 14 tag single nucleotide polymorphisms, and ophthalmological assessment was performed.RESULTSA total of 514 individuals were found to have DR. rs9640883 was significantly associated with DR (P = 0.0005). However, AKR1B1 variation was not independently associated with DR development after adjusting for relevant clinical parameters. rs9640883 was associated with duration of diabetes (P = 0.002).CONCLUSIONMany previous reports have failed to account for known risk factors for DR. The commonly reported association of AKR1B1 with DR may be due to an association of the gene with younger age at onset of diabetes.

Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase

We constructed the high-expression system of the alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli BL 21 (DE3) to characterize the enzymological and structural properties of the gene product, Alr. The Alr was expressed in the soluble fractions of the cell extract of the E. coli clone and showed alanine racemase activity. The purified Alr was a dimer with a molecular mass of 78 kDa. The Alr required pyridoxal 5'-phosphate (PLP) as a coenzyme and contained 2 mol of PLP per mol of the enzyme. The holoenzyme showed maximum absorption at 420 nm, while the reduced form of the enzyme showed it at 310 nm. The Alr was specific for alanine, and the optimum pH was observed at about nine. The Alr was relatively thermostable, and its half-life time at 60 degrees C was estimated to be 26 min. The K(m) and V(max) values were determined as follows: l-alanine to d-alanine, K(m) (l-alanine) 5.01 mM and V(max) 306 U/mg; d-alanine to l-alanine, K(m) (d-alanine) 5.24 mM and V(max) 345 U/mg. The K(eq) value was calculated to be 1.07 and showed good agreement with the theoretical value for the racemization reaction. The high substrate specificity of the Alr from C. glutamicum ATCC 13032 is expected to be a biocatalyst for d-alanine production from the l-counter part.

Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of gene products expressed in Escherichia coli

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, alanine racemases are classified into two types, monomeric and homodimeric. Alanine racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli alanine racemase-deficient mutant. From the cloning results, both strains are likely to possess only one alanine racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D-amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX alanine racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli alanine racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.

N(2)‐Substituted D,L‐Cycloserine Derivatives: Synthesis and Evaluation as Alanine Racemase Inhibitors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Molecular characterization of alanine racemase from Bifidobacterium bifidum

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties, and its peptidoglycans have the potential for applications in the fields of food science and medicine. We investigated the bifidobacterial alanine racemase, which is essential in the synthesis of d-alanine as an essential component of the peptidoglycans. Alanine racemase was purified to homogeneity from a crude extract of B. bifidum NBRC 14252. It consisted of two identical subunits with a molecular mass of 50 kDa. The enzyme required pyridoxal 5′-phosphate (PLP) as a coenzyme. The activity was lost in the presence of a thiol-modifying agent. The enzyme almost exclusively catalyzed the alanine racemization; other amino acids tested, except for serine, were inactive as substrates. The kinetic parameters of the enzyme suggested that the B. bifidum alanine racemase possesses comparatively low affinities for both the coenzyme (9.1 μM for PLP) and substrates (44.3 mM for l-alanine; 74.3 mM for d-alanine). The alr gene encoding the alanine racemase was cloned and sequenced. The alr gene complemented the d-alanine auxotrophy of Escherichia coli MB2795, and an abundant amount of the enzyme was produced in cells of the E. coli MB2795 clone. The enzymologic and kinetic properties of the purified recombinant enzyme were almost the same as those of the alanine racemase from B. bifidum NBRC 14252.

N(2)-substituted D,L-cycloserine derivatives: synthesis and evaluation as alanine racemase inhibitors.

A select series of N(2)-substituted D,L-cycloserine derivatives were prepared a ndevaluated for inhibitory activity against purified alanine racemases (alr gene product) from Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, as well as in a growth inhibition assay. N(2)-Modification led to loss of enzymatic inhibitory activity in most cases consistent with a recent proposal for cycloserine function.

The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains

The potential of the alanine racemase gene alr from Corynebacterium glutamicum ATCC 13032 to substitute for antibiotic resistance determinants in cloning systems has been investigated. The alr gene was identified by a PCR technique and its nucleotide sequence was determined. The deduced protein revealed the highest amino acid sequence similarity to the Alr protein from Mycobacterium smegmatis with 45% identical and 58% similar amino acids. A defined alr deletion mutant of C. glutamicum displayed a strict dependence on the presence of d-alanine for growth on complex and minimal medium. The alr gene was placed on a novel C. glutamicum vector which is completely free of antibiotic resistance genes. In vivo complementation of the chromosomal alr deletion with alr-carrying vectors permitted growth of the mutant strain in the absence of external d-alanine and provided strong selective pressure to maintain the plasmid. The alr gene enabled the selection of C. glutamicum transformants with a similar efficiency as the tetracycline resistance gene tetA(33). These data provided experimental evidence that the alr gene can be applied as an alternative selection marker to antibiotic resistance genes in industrial C. glutamicum strains. In an application example, the novel Δ alr host- alr + vector-system for C. glutamicum was used to overproduce the vitamin d-pantothenic acid.

Human genome search in celiac disease using gliadin cDNA as probe

Celiac disease is a wheat gliadin-promoted disorder that displays a complex genetic susceptibility associated with HLA-DQ2, and one or more unknown factor(s), possibly gliadin-like. The presence of mammalian proteins with partial gliadin similarity was suggested by transglutaminase-independent multi-tissue reactivity of gliadin-immunopurified antibodies from celiac patients. No non-plant sequence, however, was identified in gliadin peptide epitope searches of non-redundant and EST databanks via TBLASTN, BLASTP and FASTA, even at E values as high as 20. Therefore, an α-gliadin cDNA screen of human cDNA and genomic libraries was undertaken, an approach in keeping with positive human Northern and Southern analyses with the same probe. Four distinct cDNA clones were obtained, the most stringent of which (3.2 and 5.1 kb) were novel, and featured potential open reading frames with high gliadin domain II and domain IV homologies (BestFit quality scores ⩾295 and 322, respectively, versus random value 126-127). Both were also homologous to ESTs. An additional 5′ gliadin oligonucleotide screen identified the widely distributed cytoplasmic protein acyl coA hydrolase whose homology was restricted to the oligonucleotide probe (BestFit quality=215 versus 100 for random); and achaete-scute homologous protein, which displays particularly high gliadin domain II homology (BestFit quality 316 versus 111 for random). Genomic screening uncovered 16 positives, one of which was the ALR gene, whose similarity to three of gliadin’s five domains (I, II and IV; BestFit quality 322-473 versus 121-154 for random) was remarkable. More extensive was novel genomic clone 2, with fragments hybridizing to cDNA probes approximating gliadin domains I, II+IV, V and the gliadin 5′ untranslated region, and mapping by FISH to 19q13.11-13.12. Two fragments were sequenced; one was exonic, as predicted by four different programs; and test oligonucleotides suggested widespread 4 and/or 2 kb mRNA expression, even at high stringency (tm−8.8 deg. C). Taken together, it is apparent that several genes with partial gliadin homology exist in the human genome. Many bear gliadin-like T-cell epitopes, are expressed in intestine and, like transglutaminase, are cytoplasmic. Glutamine to glutamic acid or other mutation within such epitopes followed by injury or infection-related release could explain enhanced disease susceptibility in affected families.

Sequence of the putative alanine racemase operon in Staphylococcus aureus: insertional interruption of this operon reduces D-alanine substitution of lipoteichoic acid and autolysis.

A gene cluster comprising the alanine racemase gene alr was identified 5' to the sigB operon in Staphylococcus aureus. It is flanked upstream by four ORFs of which one shows similarity to the dpj gene of Escherichia coli, and downstream by two ORFs of which the last shows similarity to the E. coli pemK gene. Preliminary data suggest that the seven ORFs orf1-orf2-orf3-dpj-alr-orf6-pemK may form an operon. Disruption of the proposed operon by insertional mutagenesis leads to a drastic loss in the d-alanine (d-Ala) substitution of lipoteichoic acid and to delayed autolysis, without affecting the d-Ala substitution of the wall teichoic acid.

Overexpression of the Saccharomyces cerevisiaeMagnesium Transport System Confers Resistance to Aluminum Ion

Ionic aluminum (Al3+) is toxic to plants, microbes, fish, and animals, but the mechanism of its toxicity is unknown. We describe the isolation of two yeast genes (ALR1 and ALR2) which confer increased tolerance to Al3+ and Ga3+ ions when overexpressed while increasing strain sensitivity to Zn2+, Mn2+, Ni2+, Cu2+, Ca2+, and La3+ ions. The Alr proteins are homologous to the Salmonella typhimurium CorA protein, a bacterial Mg2+ and Co2+ transport system located in the periplasmic membrane. Yeast strains lacking ALR gene activity required additional Mg2+ for growth, and expression of either ALR1 or ALR2 corrected the Mg(2+)-requiring phenotype. The results suggest that the ALR genes encode the yeast uptake system for Mg2+ and other divalent cations. This hypothesis was supported by evidence that 57Co2+ accumulation was elevated in ALR-overexpressing strains and reduced in strains lacking ALR expression. ALR overexpression also overcame the inhibition of Co2+ uptake by Al3+ ions. The results indicate that aluminum toxicity to yeast occurs as a consequence of reduced Mg2+ influx via the Alr proteins. The molecular identification of the yeast Mg2+ transport system should lead to a better understanding of the regulation of Mg2+ homeostasis in eukaryote cells.

Structure and expression pattern of human ALR, a novel gene with strong homology to ALL-1 involved in acute leukemia and to Drosophila trithorax.

The ALL-1 gene is involved in human acute leukemia through chromosome translocations or internal rearrangements. ALL-1 is the human homologue of Drosophila trithorax. The latter is a member of the trithorax group (trx-G) genes which together with the Polycomb group (Pc-G) genes act as positive and negative regulators, respectively, to determine the body structure of Drosophila. We have cloned a novel human gene, ALR, which encodes a gigantic 5262 amino acid long protein containing a SET domain, five PHD fingers, potential zinc fingers, and a very long run of glutamines interrupted by hydrophobic residues, mostly leucine. The SET motif, PDH fingers, zinc fingers and two other regions are most similar to domains of ALL-1 and TRX. The first two motifs are also found in other trx-G and Pc-G proteins. The ALR gene was mapped to chromosome band 12q12-13, adjacent to the VDR gene. This region is involved in duplications and translocations associated with cancer. The analysis of ALR expression showed that its approximately 18 kb long mRNA is expressed, like ALL-1, in most adult tissues, including a variety of hematopoietic cells, with the exception of the liver. Whole mount in situ hybridization to early mouse embryos indicates expression in multiple tissues. Based on similarities in structure and expression pattern, ALR is likely to play a similar role to ALL-1 and trx, although its target genes have yet to be identified.

The alanine racemase gene is essential for growth of Lactobacillus plantarum.

The Lactobacillus plantarum alr gene encoding alanine racemase was cloned by complementation of an Escherichia coli Alr- DadX- double mutant strain. Knockout of the alr gene abolished all measurable alanine racemase activity, and the mutant was shown to be strictly dependent on D-alanine for growth.

The 92-min region of the Escherichia coli chromosome: location and cloning of the ubi and alr

A cosmid (pND320) bearing 42.5 kb of Escherichia coli chromosomal DNA, including the genes between xylE and ssb near minute 92 on the linkage map, was isolated by selection for complementation of a dnaB mutation. Known nucleotide (nt) sequences were used to align restriction maps in this region to the physical map of the chromosome (coordinates 4319.5 to 4362 kb), and to locate precisely and define the orientations of 19 genes. Predicted physical linkage of sequenced genes across unsequenced gaps of defined length was confirmed by the nt sequence analysis of fragments subcloned from pND320. Mutant complementation by plasmids showed that ubiA is located between malM and plsB. A previously sequenced long open reading frame that encodes the C-terminal portion of the E. coli ubiA product (4-hydroxybenzoate polyprenyltransferase, HPTase) shows a high degree of sequence identity with the corresponding segment of yeast HPTase (the COQ2 gene product). Comparison of homologous regions from E. coli and Salmonella typhimurium was used to locate precisely the gene alr that encodes alanine racemase (ARase) between dnaB and tyrB. Subcloning of alr downstream from tandem bacteriophage lambda promoters produced a plasmid that directed high-level overproduction of a soluble approx. 40-kDa protein with ARase activity.

Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles.

Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L----D and D----L directions for all three enzymes to assess the degree to which abstraction of the alpha-proton or protonation of substrate PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of alpha-3H from substrate to product and solvent exchange/substrate conversion experiments in 3H2O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.

Biosynthetic alr alanine racemase from Salmonella typhimurium: DNA and protein sequence determination

The nucleotide sequence of the alr gene encoding the biosynthetic alanine racemase in Salmonella typhimurium is reported. The sequence was determined by the dideoxy chain termination method of Sanger mostly from recombinants derived from shotgun and specific subcloning of a 2.6-kilobase region containing the alr gene. The final bridging of nonoverlapping contiguous sequences was accomplished with the use of synthetic site-specific primers. The alr gene was found to be 1077 base pairs in length encoding a protein of 359 amino acid residues. Comparison of alr with the dadB gene encoding the catabolic alanine racemase in S. typhimurium revealed almost identical size (1077 vs. 1068 base pairs) and 52% sequence identity. The respective gene products displayed 43% homology, which includes a decapeptide bearing the pyridoxal 5'-phosphate binding site.