Cloning and characterization of the hemA gene for synthesis of δ-aminolevulinic acid in Xanthomonas campestris pv. phaseoli

The gene from Xanthomonas campestris pv. phaseoli that is involved in the C5 pathway of -amino-levulinic acid (ALA) of Escherichia coli. Subcloning of deletion fragments from the initial 2.5-kilobase (kb) chromosomal fragment allowed the isolation of a 1.6-kb fragment that could complement the hemM mutation. Nucleotide sequence analysis of the 1.6-kb DNA fragment revealed an open reading frame that encodes a polypeptide of 426 amino acid residues, and the deduced molecular mass of this polypeptide is 46768 Da. The amino acid sequence shows a high degree of homology of the HemA protein, which is glutamyl-tRNA reductase, to other organisms. Thus, we examined the complementation test of the cloned gene from Xanthomonas with a hemA mutation of E. coli and found that the gene complemented the hemA mutation. These results suggest that the cloned gene is hemA and the gene from Xanthomonas also complements both hemA and hemM mutations, as in the case of the E. coli hemA. Correspondence to: Y. Murooka     

Molecular Cloning and Nucleotide Sequence of the Porphobilinogen Deaminase Gene,hemC, from Chlorobium vibrioforme

We previously reported the DNA sequence and expression of the Chlorobium vibrioforme glutamyl-tRNA reductase (hemA) gene (Majumdar et al., Arch Microbiol 156:281, 1991). The sequence downstream of the hemA gene indicated homology to Escherichia coli and Bacillus subtilis porphobilinogen deaminase (hemC) gene. The Chlorobium gene was confirmed to be the porphobilinogen deaminase gene, and complete sequence of the structural gene was obtained. A 2.8-kb DNA fragment containing the 1.3-kb hemA gene of Chlorobium was cloned into a hemC auxotroph (Sz16) of Bacillus subtilis, and complementation of the auxotroph to prototrophy was achieved. DNA sequence data showed a single open reading frame of 840 bp coding a protein of 279 amino acid residues. The deduced amino acid sequence of the Chlorobium porphobilinogen deaminase revealed 39% to 46% homology with the corresponding prokaryotic and eukaryotic sequences. Received: 20 September 1996 / Accepted: 26 October 1996     

Biosynthesis of δ-aminolevulinic acid from glutamate by Sulfolobus solfataricus

The extremely thermophilic, obligately aerobic bacterium Sulfolobus solfataricus forms the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate by the tRNA-dependent five-carbon pathway. This pathway has been previously shown to occur in plants, algae, and most prokaryotes with the exception of the -group of proteobacteria (purple bacteria). An alternative mode of ALA formation by condensation of glycine and succinyl-CoA occurs in animals, yeasts, fungi, and the -proteobacteria. Sulfolobus and several other thermophilic, sulfur-dependent bacteria, have been variously placed within a subgroup of archaea (archaebacteria) named crenarchaeotes, or have been proposed to comprise a distinct prokaryotic group designated eocytes. On the basis of ribosomal structure and certain other criteria, eocytes have been proposed as predecessors of the nuclear-cytoplasmic descent line of eukaryotes. Because aplastidic eukaryotes differ from most prokaryotes in their mode of ALA formation, and in view of the proposed affiliation of eocytes to eukaryotes, it was of interest to determine how eocytes form ALA. Sulfolobus extracts were able to incorporate label from [1-¹⁴C]glutamate, but not from [2-¹⁴C]glycine, into ALA. Glutamate incorporation was abolished by preincubation of the extract with RNase. Sulfolobus extracts contained glutamate-1-semialdehyde aminotransferase activity, which is indicative of the five-carbon pathway. Growth of Sulfolobus was inhibited by gabaculine, a mechanism-based inhibitor of glutamate-1-semialdehyde aminotransferase, an enzyme of the five-carbon ALA biosynthetic pathway. These results indicate that Sulfolobus uses the five-carbon pathway for ALA formation.Abbreviations AHA 4-amino-5-hexynoic acid - ALA -aminolevulinic acid, Gabaculine, 3-amino-2,3-dihydrobenzoic acid - GSA glutamate 1-semialdehyde     

Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, -aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO₂. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA^Glu, ATP, Mg²⁺, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[³H]glutamate or 1-[¹⁴C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[¹⁴C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the subgroup of purple bacteria.Abbreviations ALA -aminolevulinic acid - DTT dithiothreitol - PALP pyridoxal phosphate - SDS sodium dodecyl sulfate - tricine N-tris-(hydroxymethyl)methylglycine     

Cloning and sequencing of the hemA gene of Rhodobacter capsulatus and isolation of a δ-aminolevulinic acid-dependent mutant strain

Summary The Rhodobacter capsulatus hemA gene, coding for the enzyme -aminolevulinic acid synthase (ALAS), was isolated from a genome bank by hybridization with a hemT probe from Rhodobacter sphaeroides. Subcloning of the initial 3.9 kb HindIII fragment allowed the isolation of a 2.5 kb HindIII-BglII fragment which was able to complement the -aminolevulinic acid-requiring (ALA-requiring) Escherichia coli mutant SHSP19. DNA sequencing revealed an open reading frame coding for a protein with 401 amino acids which displayed similarity to the amino acid sequences of other known ALASs. However, no resemblance was seen to the HemA protein of E. coli K12. Based on the sequence data, an ALA-requiring mutant strain of R. capsulatus was constructed by site-directed insertion mutagenesis. Introduction of a plasmid, containing the hemA gene of R. capsulatus on the 3.9 kb HindIII fragment, restored ALA-independent growth of the mutant indicating that there is only one gene for ALA biosynthesis in R. capsulatus. Transfer of the R factor pRPS404 and hybridization analysis revealed that the ALAS gene is not located within the major photosynthetic gene cluster.Part of this research was presented at the Symposium on Molecular Biology of Membrane-Bound Complexes in Phototrophic Bacteria, Freiburg, FRG, 2–5 August 1989     

Heme-deficient mutants of Salmonella typhimurium: two genes required for ALA synthesis

Summary The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). We have isolated, mapped and characterized a large number of Salmonella typhimurium mutants auxotrophic for ALA. These mutants carry defects in either one of two genes, both required for ALA synthesis. The previously identified hemA gene maps at 35 min, and the hemL gene maps at 5 min on the S. typhimurium genetic map. Mutants in hemA and hemL are defective for aerobic and anaerobic respiration, and appear to be oxygen sensitive. The Hem^– phenotype of hemL mutants is less severe than that of hemA mutants. Although hemA and hemL mutants are deficient in heme synthesis, genetic tests indicate that they still synthesize two minor products of the heme pathway, siroheme and cobalamin (vitamin B₁₂), under anaerobic conditions. In contrast, hemB, hemC and cysG mutants, blocked after ALA synthesis, make neither siroheme nor vitamin B₁₂. Double mutants defective in both hemA and hemL also make siroheme. We suggest that hemA and hemL are required for one route of ALA synthesis and that a second, minor route of ALA synthesis may operate in S. typhimurium; this second pathway would be independent of the hemA and hemL functions.Abbreviations Amp ampicillin - Cam chloramphenicol - Kan kanamycin - Tet tetracycline - Str streptomycin - X-gal 5-bromo-4-chloro-3-indolyl--d-galactoside - DES diethyl sulfate     

The <Emphasis Type="Italic">Chlamydomonas reinhardtii gtr </Emphasis>Gene Encoding the Tetrapyrrole Biosynthetic Enzyme Glutamyl-tRNA Reductase: Structure of the Gene and Properties of the Expressed Enzyme

Plants, algae, cyanobacteria and many other bacteria synthesize the tetrapyrrole precursor, δ-aminolevulinic acid (ALA), from glutamate by means of a tRNA^Glu-mediated pathway. The enzyme glutamyl-tRNA reductase (GTR) catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. Chlamydomonas reinhardtii mRNA encoding gtr was sequenced from a cDNA and genomic libraries. The 3179-bp gtr cDNA contains a 1566-bp open reading frame that encodes a 522-amino acid polypeptide. After removal of the predicted transit peptide, the mature 480-residue GTR has a calculated molecular weight of 52,502. The deduced C. reinhardtii mature GTR amino acid sequence has more than 55% identity to a GTR sequence of Arabidopsis thaliana, and significant similarity to GTR proteins of other plants and prokaryotes. Southern blot analysis of C. reinhardtii genomic DNA indicates that C. reinhardtii has only one gtr gene. Genomic DNA sequencing revealed the presence of a small intron near the putative transit peptide cleavage site. Expression constructs for the full-length initial gtr translation product, the mature protein after transit peptide removal, and the coding sequence of the second exon were cloned into expression vector that also introduced a C-terminal His₆ tag. All of these constructs were expressed in E. coli, and both the mature protein and the exon 2 translation product complemented a hemA mutation. The expressed proteins were purified by Ni-affinity column chromatography to yield active GTR. Purified mature GTR was not inhibited by heme, but heme inhibition was restored upon addition of C. reinhardtii soluble proteins.     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.     

Members of the pogo superfamily of DNA-mediated transposons in the human genome

Bacillus subtilis, likeEscherichia coli, possesses several sets of genes involved in the utilization of-glucosides. InE. coli, all these genes are cryptic, including the genes forming thebgl operon, thus leading to a Bgl^– phenotype. We screened forB. subtilis chromosomal DNA fragments capable of reverting the Bgl⁺ phenotype associated with anE. coli hns mutant to the Bgl^– wild-type phenotype. OneB. subtilis chromosomal fragment having this property was selected. It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from thebglP gene. Deletion studies as well as subcloning experiments allowed us to prove that the putativeB. subtilis bglP RAT sequence was responsible for the repression of theE. coli bgl operon. We propose that this repression results from the titration of the BglG antiterminator protein ofE. coli bgl operon by our putativeB. subtilis bglP RAT sequence. Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs.     

A gene cluster inChlorobium vibrioforme encoding the first enzymes of chlorophyll biosynthesis

A cloned 5.8-kb genomic fragment of the green sulfur bacteriumChlorobium vibrioforme encodes the genes for three enzymes catalyzing early steps in the biosynthetic pathway of tetrapyrroles, common to chlorophyll and heme. ThehemA, hemC andhemD genes encode the enzymes glutamyl tRNA dehydrogenase, porphobilinogen deaminase and uroporphyrinogen III synthase, respectively. The cloned genes were expressed in transformedEscherichia coli orSalmonella typhimurium and conferred autotrophy on the respective auxotrophs. Activities of the enzymes encoded by the cloned genes were demonstrated in vitro, with cell extracts obtained from the transformed enterobacteria. The proximity of these genes indicates that they form a cluster inChlorobium vibrioforme, while in most other organisms they appear to be scattered. The presence of this cluster may imply coordinate regulation of the genes involved and they may constitute an operon.     

Cloning of an Arabidopsis thaliana cDNA encoding cystathionine β-lyase by functional complementation in Escherichia coli

Cystathionine -lyase, the second enzyme involved in the methionine biosynthetic pathway in plants, catalyses the synthesis of homocysteine from cystathionine. A cDNA encoding cystathionine -lyase was cloned from an Arabidopsis thaliana expression library by complementation of an Escherichia coli mutant deficient in this enzyme. As deduced from the full-length nucleotide sequence (1.7 kb), the polypeptide contains 464 amino acids and presents a predicted M _r of 50372. A. thaliana cystathionine -lyase exhibits 22% sequence identity with the E. coli corresponding enzyme and contains a 70 amino acid N-terminal additional sequence compared with the bacterial protein. Since the general features of chloroplast transit peptides could be observed in this amino-terminal extension, we propose a chloroplast localization for the cDNA-encoded enzyme. Southern blot analysis suggested that cystathionine -lyase is encoded by a single copy gene in A. thaliana.     

The nucleotide sequence of the lipo-penicillinase gene of alkalophilic Bacillus sp. strain 170

The lipo-penicillinase (LIPEN) gene from an alkalophilic Bacillus sp. strain 170 was cloned in Escherichia coli using the vector pHSG399. A plasmid, pFAP121, was isolated from an ampicillin resistant transformant and the cloned LIPEN gene was found to be in a 2.2 kb DNA fragment. The nucleotide sequence of a 1.9 kb segment encoding the LIPEN was determined. This segment showed an open reading frame which would encode a polypeptide of 310 amino acids. The amino acid sequence of this LIPEN gene product has strong homology with those of the Bacillus cereus -lactamase III and Bacillus licheniformis penicillinase.     

Close genetic relationship between Nitrobacter hamburgensis nitrite oxidoreductase and Escherichia coli nitrate reductases

The nitrite oxidoreductase (NOR) from the facultative nitrite-oxidizing bacterium Nitrobacter hamburgensis X14 was investigated genetically. In order to develop a probe for the gene norB, the N-terminal amino acid sequence of the NOR -subunit (NorB) was determined. Based on that amino acid sequence, an oligo-nucleotide was derived that was used for the identification and cloning of gene norB. Sequence analysis of DNA fragments revealed three adjacent open reading frames in the order norA, norX, norB. The DNA sequences of norX and norB represented complete genes while the open reading frame of norA was truncated by the cloning site. The deduced amino acid sequence of protein NorB contained four cysteine clusters with striking homology to those of iron-sulfur centers of bacterial ferredoxins. NorB shares significant sequence similarity to the -subunits (NarH, NarY) of the two dissimilatory nitrate reductases (NRA, NRZ) of Escherichia coli. Additionally, the derived amino acid sequence of the truncated open reading frame of norA showed striking resemblance to the -subunits (NarG, NarZ) of the E. coli nitrate reductases.     

Protein export elements from Lactococcus lactis

Summary Broad-host-range plasmids carrying -amylase or -lactamase reporter genes lacking a signal sequence were used to select export elements from Lactococcus lactis chromosomal DNA that could function as signal sequences. Fragments containing such elements were identified by their ability to direct the export of the reporter proteins in Escherichia coli. Several of the selected export elements were also active in Bacillus subtilis and L. lactis, although the efficiencies depended strongly on the host organism and reporter gene used. The export elements AL9 and BL1 were highly efficient in L. lactis in the expression and secretion of at least two heterologous proteins (Bacillus licheniformis -amylase and E. coli TEM--lactamase). AL9 even permitted growth of this organism on starch as the sole carbon source. Nucleotide sequence analysis of five selected fragments indicated that these encode oligopeptides with the major characteristics of typical signal peptides. The putative expression signals had a limited similarity to previously described expression signals for E. coli, B. subtilis and L. lactis. Differences in both expression and export efficiency are likely to underlie the host-specific effects.     

Cloning of hemA from Rhizobium sp. NGR234 and symbiotic phenotype of a gene-directed mutant in diverse legume genera

Summary The hemA gene which encodes -aminolaevulinic acid synthase (ALAS), was cloned and characterized from the broad host-range Rhizobium strain NGR234. A cosmid, identified by hybridization with the cloned gene of R. meliloti and complementation of an R. meliloti hemA mutant, was subcloned to yield a 5.5 kb fragment containing the entire NGR234 gene. A physical-genetic map was made and the interposon was introduced into a single EcoRI site which bisects the gene. The mutated gene was homogenotized into NGR234 to generate a hemA mutant, with a view to evaluating the role of rhizobial bacteroid ALAS activity for a wide variety of legume symbioses. The mutant strain formed an ineffective (Fix^–) symbiosis with all tested host plants. These included tropical legumes that produce either indeterminate (Leucaena) or determinate (Desmodium, Macroptilium, Lablab, Vigna) root nodules.Abbreviations ALA -aminolaevulinic acid - ALAS aminolaevulinic acid synthase - Lb leghaemoglobin - Lb-haem haem moiety of leghaemoglobin     

Molecular cloning and characterization of RGA1 encoding a G protein α subunit from rice (Oryza sativa L. IR-36)

A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library prepared from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 M [adenylate-³²P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.     

The border fresidues of the dihydrofolate reductase domain inEscherichia coli β-galactosidase correspond to the positions of introns 1 and 5 of dihydrofolate reductase of chicken

Summary In-galactosidase ofEscherichia coli residues 820–934 are similar to residues in dihydrofolate reductase ofE. coli. Dihydrofolate reductase ofE. coli and chicken are also similar and have identical tertiary structures. I used the similarity of the three-dimensional structure of prokaryotic and eukaryotic dihydrofolage reductases to align the chicken dihydrofolate reductase and the similar residues of-galactosidase. The positions of introns 1 and 5 of the chicken dihydrofolate reductase gene correspond exactly to the start and the end of the dihydrofolate reductase-like domain in the-galactosidase polypeptide chain. This equivalence of intron positions in a eukaryotic gene and domain structure in a prokaryotic protein was interpreted as evidence for a common origin of both genes.     

Cloning and characterization of the bifunctional alcohol/acetaldehyde dehydrogenase gene (<Emphasis Type="BoldItalic">adhE</Emphasis>) in <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> isolated from kimchi

A bifunctional alcohol/acetaldehyde dehydrogenase (AdhE) gene (adhE) was cloned from Leuconostoc mesenteroides C7 (LMC7), which is the dominant lactic acid bacterium produced during heterofermentation of kimchi. The nucleotide sequence of the DNA fragment containing putative adhE, which is 2685 bp long and encodes an 886 amino acid polypeptide, exhibits 99% homology with Leu. mesenteroides sp. cremoris. The deduced AdhE comprises two conserved domains: alcohol dehydrogenase (Adh) and acetaldehyde dehydrogenase (Aldh). Moreover, two NAD-binding sites were observed, based on the presence of the GXGXXG motif. A pADHE containing the adhE gene expressed AdhE at the translational level in Escherichia coli BL21, which was at a higher level than in E. coli DH5 and E. coli JM109. The AdhE of LMC7 showed Adh and Aldh activities that, when expressed in E. coli. BL21, were 7.5 and 5.7 U mg^-1 , respectively.     

Characterization of two genes encoding small heat-shock proteins in Arabidopsis thaliana

Summary Using the technique of differential hybridization screening, we have isolated the cDNAs for two low-molecular-mass heat-shock proteins and their corresponding genes, HSP17.4 and HSP18.2, from Arabidopsis thaliana. These two genes encode polypeptides that are 79.2% identical to each other with respect to amino acid sequence, and contain several overlapping sequences that are similar to the consensus sequences for the heat-shock elements (HSE) in Drosophila in the regions upstream from the promoters. The 5 region of the HSP18.2 gene has been fused, in frame, to the uidA gene from Escherichia coli which encodes -glucuronidase (GUS), and the product has been introduced into petunia by Agrobacterium-mediated transformation. We have demonstrated that the GUS activity in transformed petunia plants is enhanced by heat shock.