Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.     

Glutamyl-tRNA reductase from Escherichia coli and Synechocystis 6803. Gene structure and expression.

In the cyanobacterium Synechocystis sp. PCC 6803 and in the enterobacterium Escherichia coli delta-amino-levulinic acid (ALA) is formed from glutamyl-tRNA by the sequential action of two enzymes, glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase. E. coli has two GluTR proteins with sizes of 45 kDa (GluTR45) and 85 kDa (GluTR85) (Jahn, D., Michelsen, U., and S?ll, D. (1991) J. Biol. Chem. 266, 2542-2548). The hemA gene, isolated from E. coli and several other eubacteria, has been proposed to encode a structural component of GluTR. Because of the inability to overexpress this gene in E. coli, we demonstrate directly GluTR function for the E. coli hemA gene product by its expression and functional analysis in yeast, which does not form ALA from Glu-tRNA. Gel filtration experiments demonstrated definitively that the yeast-expressed HemA protein corresponded to GluTR45. Furthermore, analysis of GluTR activity in an E. coli strain with a disrupted hemA gene displayed GluTR85, but not GluTR45 activity. The hemA gene from Synechocystis 6803 was cloned by functional complementation in E. coli. DNA sequence analysis revealed an open reading frame capable of encoding a 427-amino acid polypeptide (molecular mass of 47,525 Da). The Synechocystis 6803 amino acid sequence shows significant similarity upon alignment with HemA sequences from E. coli, Bacillus subtilis, Salmonella typhimurium, and Chlorobium vibrioforme but does not contain the amino acid sequence derived from the N terminus of the previously purified GluTR protein (Rieble, S., and Beale, S. I. (1991) J. Biol. Chem. 266, 9740-9745). These experiments are the first direct demonstration of GluTR activity of the HemA protein and provide further evidence for two pathways of ALA formation in prokaryotes.     

Cloning and characterization of genes involved in the biosynthesis of delta-aminolevulinic acid in Escherichia coli.

Several mutants of Escherichia coli that had lost their ability to synthesize delta-aminolevulinic acid (ALA) via the C5 pathway were isolated. Their defective loci were classified into two groups, AlaA- and AlaB-. The genes that complemented these mutations were cloned. Nucleotide sequencing indicated that the gene that complemented AlaA- was identical to hemL which is located at 4 min on the E. coli chromosome and encodes glutamate 1-semialdehyde aminotransferase. The gene complementing AlaB- contained an open reading frame (ORF) encoding a polypeptide of 207 amino acids that was found to be a new gene involved in the synthesis of ALA via the C5 pathway. Thus, we designated the gene hemM. The hemM gene was adjacent to hemA that is located at 27 min and previously thought to encode glutamyl-tRNA dehydrogenase. However, we found that hemA complemented both the AlaA- (hemL) and AlaB- (hemM) mutants defective in the C5 pathway although the transformants showed small colonies on the selective medium without ALA. These results suggest that hemA is not involved in the C5 pathway, but controls a second, minor pathway for the synthesis of ALA.     

Cloning of the argF gene encoding the ornithine carbamoyltransferase from Corynebacterium glutamicum.

The argF gene encoding ornithine carbamoyl-transferase (OTCase; EC2.1.3.3) has been cloned from Corynebacterium glutamicum by transforming the Escherichia coli arginine auxotroph with the genomic DNA library. The cloned DNA also complements the E. coli argG mutant, suggesting a clustered organization of the genes in the genome. We have determined the DNA sequence of the minimal fragment complementing the E. coli argF mutant. The coding region of the cloned gene is 957 nucleotides long with a deduced molecular mass of about 35 kDa polypeptide. The enzyme activity and size of the expressed protein in the E. coli auxotroph carrying the argF gene revealed that the cloned gene indeed codes for OTCase. Analysis of the amino acid sequence of the predicted protein revealed a strong similarity to the corresponding protein of other bacteria.     

Cloning and sequencing of the gene encoding a 125-kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene.

Using the vector pGEM-4-blue, a 4,251-base-pair DNA fragment containing the gene for the surface (S)-layer protein of Bacillus sphaericus 2362 was cloned into Escherichia coli. Determination of the nucleotide sequence indicated an open reading frame (ORF) coding for a protein of 1,176 amino acids with a molecular size of 125 kilodaltons (kDa). A protein of this size which reacted with antibody to the 122-kDa S-layer protein of B. sphaericus was detected in cells of E. coli containing the recombinant plasmid. Analysis of the deduced amino acid sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The first amino acid of the N-terminal sequence of the 122-kDa S-layer protein followed the predicted cleavage site of the leader peptide in the 125-kDa protein. A sequence characteristic of promoters expressed during vegetative growth was found within a 177-base-pair region upstream from the ORF coding for the 125-kDa protein. This putative promoter may account for the expression of this gene during the vegetative growth of B. sphaericus and E. coli. The gene for the 125-kDa protein was followed by an inverted repeat characteristic of terminators. Downstream from this gene (11.2 kilobases) was an ORF coding for a putative 80-kDa protein having a high sequence similarity to the 125-kDa protein. Evidence was presented indicating that this gene is cryptic.     

Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.

The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). Mutations in two genes, hemA and hemL, result in auxotrophy for ALA in Salmonella typhimurium, but the roles played by these genes and the mechanism of ALA synthesis are not understood. I have cloned and sequenced the S. typhimurium hemA gene. The predicted polypeptide sequence for the HemA protein shows no similarity to known ALA synthases, and no ALA synthase activity was detected in extracts prepared from strains carrying the cloned hemA gene. Genetic analysis, DNA sequencing of amber mutations, and maxicell studies proved that the open reading frame identified in the DNA sequence encodes HemA. Another surprising finding of this study is that hemA lies directly upstream of prfA, which encodes peptide chain release factor 1 (RF-1). A hemA::Kan insertion mutation, constructed in vitro, was transferred to the chromosome and used to show that these two genes form an operon. The hemA gene ends with an amber codon, recognized by RF-1. I suggest a model for autogenous control of prfA expression by translation reinitiation.     

重组Rhodobacter sphaeroides hemA表达的调控

RhodobactersphaeroideshemA编码5氨基乙酰丙酸合酶(ALAS),催化磷酸吡哆醛依赖性琥珀酰CoA和甘氨酸缩合成ALA.将R.spaeroideshemA导入E.coli进行表达,当hemA具有与lac启动子相同的转录方向时,ALAS有活性.lac启动子与hemA之间的距离会影响ALAS在不同培养基上的表达.E.coli宿主菌对ALAS表达、ALA产量有显著影响,在实验所用6种菌株中,E.coliDH1是最佳宿主菌(P<0.05).ALAS表达还与碳源有关,琥珀酸为碳源时,重组ALAS活性最高(P<0.05),以乳酸为碳源时,ALAS活性很低.重组ALAS活性也受培养基pH值影响,pH6.5时,活性最高(P<0.05).     

5-Aminolevulinic acid synthesis in Escherichia coli.

A hemA mutant of Escherichia coli containing a multicopy plasmid which complemented the mutation excreted 5-aminolevulinic acid (ALA) into the medium. [1-14C]glutamate was substantially incorporated into ALA by this strain, whereas [2-14C]glycine was not. Periodate degradation of labeled ALA showed that C-5 of ALA was derived from C-1 of glutamate. The synthesis of ALA by two sonicate fractions which had been processed by gel filtration and dialysis, respectively, was dependent on glutamate, ATP, NADPH, tRNA(Glu), and pyridoxal phosphate. tRNA(Glu) stimulated ALA synthesis in a concentration-dependent manner. Pretreatment with RNase reduced this stimulation. The amino acid sequence of the cloned insert, derived from the nucleotide sequence (J.-M. Li, C. S. Russell, and S. D. Cosloy, J. Cell Biol. 107:617a, 1988), showed no homology with any ALA synthase sequenced to date. These results suggest that E. coli synthesizes ALA by the C5 pathway from the intact five-carbon chain of glutamate.     

表达浑球红细菌hemA基因生产5-氨基乙酰丙酸的研究

5-氨基乙酰丙酸（5-aminolevulinate acid,ALA）在农业,工业,医药业具有广泛的应用。ALA由5-氨基乙酰丙酸合酶（5-aminolevulinate acid synthase, ALAS）催化产生,其生物合成受终产物血红素的反馈抑制。本研究克隆一种浑球红细菌的hemA基因,序列分析其与已报道的基因具有96％的同源性,蛋白质编码区域也发生改变,并利用生物信息学软件进行同源关系的分析。采用大肠杆菌重组技术,构建表达载体pET28a—hemA,表达了有活性的浑球红细菌（Rhodobacter sphaeroides）的ALAS,研究了IPTG诱导和PH对研究ALAS的影响,同时分析了重组菌株合成ALA的能力,测定胞外产量。结果表明,在PH6．5,30mmol／L琥珀酸和60mmol／L甘氨酸培养条件下,胞外ALA的最大合成量达到669mg／L。     

Cloning and sequencing of some genes responsible for porphyrin biosynthesis from the anaerobic bacterium Clostridium josui.

The 6.2-kbp DNA fragment encoding the enzymes in the porphyrin synthesis pathway of a cellulolytic anaerobe, Clostridium josui, was cloned into Escherichia coli and sequenced. This fragment contained four hem genes, hemA, hemC, hemD, and hemB, in order, which were homologous to the corresponding genes from E. coli and Bacillus subtilis. A typical promoter sequence was found only upstream of hemA, suggesting that these four genes were under the control of this promoter as an operon. The hemA and hemD genes cloned from C. josui were able to complement the hemA and hemD mutations, respectively, of E. coli. The COOH-terminal region of C. josui HemA and the NH2-terminal region of C. josui HemD were homologous to E. coli CysG (Met-1 to Leu-151) and to E. coli CysG (Asp-213 to Phe-454) and Pseudomonas denitrificans CobA, respectively. Furthermore, the cloned 6.2-kbp DNA fragment complemented E. coli cysG mutants. These results suggested that both C. josui hemA and hemD encode bifunctional enzymes.     

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: effect of a transposon insertion in the hbdA gene

The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in Rhodobacter sphaeroides its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in R. sphaeroides 2.4.1: hemA and hemT. In our investigations of hemA regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which hemA expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985-993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of hbdA, coding for S-(+)-beta-hydroxybutyryl-coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the hbdA gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of hemA expression.     

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning the gyrA gene of Bacillus subtilis.

We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O. The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation. Functional gyrA activity encoded by this fragment complements E. coli gyrA mutants. Recombination between the Bacillus sequences and the E. coli chromosome did not occur. The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E. coli as is the E. coli gene. The cloned DNA precisely defines the physical location of the gyrA mutation on the B. subtilis chromosome. Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B. subtilis to nalidixic acid resistance, both alleles have been cloned.     

Expression of adenovirus type 12 E1b 58-kDa protein in Escherichia coli and production of antibodies raised against a 58-kDa::beta-galactosidase fusion protein

DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector. The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal. Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein. Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells. Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression. We have not been able to detect substantial amounts of the 58-kDa protein in these cells. However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E. coli.     

Nucleotide sequence of mouse 5-aminolevulinic acid synthase cDNA and expression of its gene in hepatic and erythroid tissues

The cDNA coding for 5-aminolevulinic acid (ALA) synthase (EC 2.3.1.37) in both liver and anemic spleen of the mouse has been cloned. The liver clone was selected by complementation of an Escherichia coli hemA mutant. Erythroid clones were obtained by screening a cDNA library made from mouse anemic spleen RNA, using the liver cDNA as a probe. The sequences of the spleen-derived and liver-derived cDNAs are identical. The nucleotide sequence and predicted amino acid (aa) sequence of a 1.85-kb spleen-derived cDNA is presented. The mouse ALA synthase as sequence displays extensive homology to ALA synthase of chick embryonic liver. The ALA synthase mRNA, detected by Northern blot analysis, was the same size, approx. 2.3 kb, in mouse liver, anemic spleen, and mouse erythroleukemia cells. It is therefore unlikely that different isozymic forms of ALA synthase are present in mouse erythroid and hepatic tissue and this is not the basis for the different effects of heme and porphyrinogenic compounds on the expression of liver and erythroid ALA synthase.     

Hemin-Deficient Mutants of Salmonella typhimurium

Nine hemin-deficient mutants of Salmonella typhimurium LT2 were isolated as neomycin-resistant colonies. Five of these mutants could be stimulated by Delta-aminolevulinic acid (Delta-ALA), thus representing hemA mutants. Since S. typhimurium LT2 is not able to incorporate hemin, the identification of the mutants not stimulated by Delta-ALA was made on the basis of the simultaneous loss of catalase activity and cytochromes. The hemA gene was mapped by conjugation in the trp region, probably in the order purB-pyrD-hemA-trp; the episome FT(71)trp does not carry the hemA gene. Transductional intercrosses by phage P22 indicate that hemA 11, 12, 13, and 37 are at very closely linked sites, whereas hemA14 is at a more distant site in the same or an adjacent gene. No joint transduction was detected between hemA and trp or pyrF. The loci affected in the other hemin-deficient mutants were linked in conjugation to the pro(+) marker (frequency of linkage, 88 to 97%), but cotransduction of the two markers could not be obtained. The episome F lac hem purE, which originates from Escherichia coli K-12, could complement these hemin-deficient mutants of S. typhimurium LT2. As a result, the sequence of the markers on the chromosome of S. typhimurium LT2 is probably pro heme purE, analogous to the sequence found in E. coli K-12. Thus, the chromosome of S. typhimurium also possesses two hem regions, with a location similar to that described in E. coli K-12.