hemA Gene Research Articles

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 Söll, 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.

Glutamyl-tRNA reductase activity in Bacillus subtilis is dependent on the hemA gene product.

The Bacillus subtilis hemAXCDBL operon encodes enzymes for the synthesis of 5-aminolaevuline acid via the C5 pathway (hemA and hemL) and uroporphyrinogen III (hemB, hemC and hemD). B. subtilis HemA protein (molecular mass 50 kDa) was overexpressed in hemA mutant of both Escherichia coli and B. subtilis. A mutant B. subtilis HemA protein with a Cys to Tyr change at position 105 was also overexpressed. Both wild-type and mutant HemA proteins migrated as oligomers (molecular mass greater than or equal to 230 kDa) on gel-filtration columns. All column fractions containing wild-type HemA protein had glutamyl-tRNA reductase activity. No glutamyl-tRNA reductase activity was found with the mutant HemA protein. It is concluded that the B. subtilis hemA gene product is identical to, or part of, the glutamyl-tRNA reductase of the C5 pathway.

Structure and expression of the Chlorobium vibrioforme hemA gene

The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, delta-aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway. A 1.9-kb clone of genomic DNA from C. vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced. The transforming C. vibrioforme DNA has significant sequence similarity to the E. coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174. This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E. coli, S. typhimurium, and B. subtilis hemA genes. No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S. typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase. These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway. A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E. coli and B. subtilis hemC genes. This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E. coli and B. subtilis HemC peptides. hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen tension regulated expression of the hemA gene of Rhodobacter capsulatus

The promoter of the Rhodobacter capsulatus hemA gene, coding for the enzyme δ-aminolevulinic acid synthase (ALAS), was identified by trans-complementation of a δ-aminolevulinic acid (ALA)-dependent mutant and found to be located within a 170 bp region proximal to the hemA gene. The activity of the hemA promoter was demonstrated by lacZ fusion and in vitro transcription-translation. An open reading frame (ORFX) was found downstream of hemA. The activity of the hemA promoter, but not that of the ORFX promoter, increased when oxygen tension was lowered in the culture. Deletions upstream of the hemA promoter region did not affect ALAS activity and formation of pigment-protein complexes in R. capsulatus.

Common cis-acting region responsible for transcriptional regulation of Bradyrhizobium japonicum hydrogenase by nickel, oxygen, and hydrogen

Bradyrhizobium japonicum expresses hydrogenase in microaerophilic free-living conditions in the presence of nickel. Plasmid-borne hup-lacZ transcriptional fusion constructs were used to study the regulation of the hydrogenase gene. The hydrogenase gene was transcriptionally induced under microaerobic conditions (0.1 to 3.0% partial pressure O2). The hydrogenase gene was not transcribed or was poorly transcribed in strictly anaerobic conditions or conditions above 3.0% O2. Hydrogen gas at levels as low as 0.1% partial pressure induced hydrogenase transcription, and a high level of transcription was maintained up to at least 10% H2 concentration. No transcription was observed in the absence of H2. Hydrogenase was regulated by H2, O2, and Ni when the 5'-upstream sequence was pared down to include base number -168. However, when the upstream sequence was pared down to base number -118, the regulatory response to O2, H2, and Ni levels was negated. Thus, a common cis-acting regulatory region localized within 50 bp is critical for the regulation of hydrogenase by hydrogen, oxygen, and nickel. As a control, the B. japonicum hemA gene which codes for delta-aminolevulinic acid synthase was also fused to the promoterless lacZ gene, and its regulation was tested in the presence of various concentrations of O2 and H2. hemA-lacZ transcription was not dependent on levels of Ni, O2, or H2. Two different hup-lacZ fusions were tested in a Hup- background, strain JH47; these hup-lacZ constructs in JH47 demonstrated dependency on nickel, O2, and H2, indicating that the hydrogenase protein itself is not a sensor for regulation by O2, H2, or nickel.

Cloning and nucleotide sequence of the hemA gene of Agrobacterium radiobacter

The hemA gene of Agrobacterium radiobacter ATCC4718 was identified by hybridization with a hemA probe from Rhizobium meliloti and cloned by complementation of a hemA mutant of Escherichia coli K12. E. coli hemA transformants carrying the hemA gene of Agrobacterium showed delta-aminolevulinic acid synthetase (delta-ALAS) activity in vitro. The hemA gene was carried on a 4.4 kb EcoRI fragment which could be reduced to a 2.6 kb EcoRI-SstI fragment without affecting its complementing or delta-ALAS activity. The sequence of the hemA gene showed an open reading frame of 1215 nucleotides, which could code for a protein of 44,361 Da. This is very close to the molecular weight of the HemA protein obtained using an in vitro coupled transcription-translation system (45,000 Da). Comparison of amino acid sequences of the delta-ALAS of A. radiobacter and Bradyrhizobium japonicum showed strong homology between the two enzymes; less, but still significant, homology was observed when A. radiobacter and human delta-ALAS were compared. Primer extension experiments enabled us to identify two promoters for the hemA gene of A. radiobacter. One of these promoters shows some similarity to the first promoter of the hemA gene of R. meliloti.

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III.

We have recently reported (M. Petricek, L. Rutberg, I. Schröder, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.

Use of a lacZ fusion to study transcriptional regulation of the Rhodobacter capsulatus hemA gene

An EcoRI fragment containing the Rhodobacter capsulatus hemA promoter has been cloned into a lacZ translational fusion vector. The resulting plasmid produced a hemA-lacZ fusion protein with a molecular mass of 147,000. Expression of the hemA-lacZ fusion, as measured by production of beta-galactosidase, was regulated 2- to 3-fold by oxygen tension. The unexpectedly small change in beta-galactosidase levels suggests that transcriptional regulation of the hemA gene is not the major factor in oxygen-mediated control of porphyrin synthesis.

Use of alacZfusion to study transcriptional regulation of theRhodobacter capsulatus hemAgene

An EcoRI fragment containing the Rhodobacter capsulatus hemA promoter has been cloned into a lacZ translational fusion vector. The resulting plasmid produced a hemA-lacZ fusion protein with a molecular mass of 147,000. Expression of the hemA-lacZ fusion, as measured by production of beta-galactosidase, was regulated 2- to 3-fold by oxygen tension. The unexpectedly small change in beta-galactosidase levels suggests that transcriptional regulation of the hemA gene is not the major factor in oxygen-mediated control of porphyrin synthesis.

Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.

Isolation and characterization of a new mutant of Saccharomyces cerevisiae with altered synthesis of 5-aminolevulinic acid

A new gene, RHM1, required for normal production of 5-aminolevulinic acid by Saccharomyces cerevisiae, was identified by a novel screening method. Ethyl methanesulfonate treatment of a fluorescent porphyric strain bearing the pop3-1 mutation produced nonfluorescent or weakly fluorescent mutants with defects in early stages of tetrapyrrole biosynthesis. Class I mutants defective in synthesis of 5-aminolevulinate regained fluorescence when grown on medium supplemented with 5-aminolevulinate, whereas class II mutants altered in later biosynthetic steps did not. Among six recessive class I mutants, at least three complementation groups were found. One mutant contained an allele of HEM1, the structural gene for 5-aminolevulinate synthase, and two mutants contained alleles of the regulatory gene CYC4. The remaining mutants contained genes complementary to both hem1 and cyc4. Mutant strain DA3-RS3/68 contained mutant gene rhm1, which segregated independently of hem1 and cyc4 during meiosis. 5-Aminolevulinate synthase activity of the rhm1 mutant was 35 to 40% of that of the parental pop3-1 strain, whereas intracellular 5-aminolevulinate concentration was only 3 to 4% of the parental value. Transformation of an rhm1 strain with a multicopy plasmid containing the cloned HEM1 gene restored normal levels of 5-aminolevulinate synthase activity, but intracellular 5-aminolevulinate was increased to only 9 to 10% of normal. We concluded that RHM1 could control either targeting of 5-aminolevulinate synthase to the mitochondrial matrix or the activity of the enzyme in vivo.

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

The Rhodobacter capsulatus hemA gene, coding for the enzyme delta-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 delta-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.

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.

Cloning and structure of the hemA gene of Escherichia coli K-12

An Escherichia coli gene, which complements two independent hemA mutants of E. coli, has been cloned onto a multi-copy plasmid and both its strands have been sequenced. Both complemented mutants produce 5-aminolevulinic acid (ALA) and display fluorescence after 24 h. The cloned sequence appears to encode a 46-kDa protein, which when produced in the maxicell procedure is processed to a 41-kDa protein as determined by sodium dodecyi sulfate-polyacrylamide-gel electrophoresis. The amino acid sequence of the cloned gene product shows no significant homologies with any cloned ALA synthase, nor with any protein, in two E. coli databanks. A second cloned gene fragment, which has its coding region 34 bp away from the coding region of the gene that complements hemA, has been identified as part of protein release factor l (RF1), thus confirming the location of hemA at min 26.7 and mapping it precisely near RF1. We have shown that E. coli utilizes the intact five-carbon chain of glutamate for the synthesis of ALA.

Isolation, nucleotide sequence, and preliminary characterization of the Escherichia coli K-12 hemA gene

The Escherichia coli hemA gene, essential for the synthesis of 5-aminolevulinic acid (ALA), was isolated and sequenced. The following criteria identified the cloned gene as hemA. (i) The gene complemented a hemA mutation of E. coli. (ii) The gene was localized to approximately 26.7 min on the E. coli chromosomal linkage map, consistent with the location of the mapped hemA locus. Furthermore, DNA sequence analysis established that the cloned gene lay directly upstream of prfA, which encodes polypeptide chain release factor 1. (iii) Deletion of the gene resulted in a concomitant requirement for ALA. The hemA gene directed the synthesis of a 46-kilodalton polypeptide in maxicell experiments, as predicted by the coding sequence. The DNA and deduced amino acid sequences of the E. coli hemA gene displayed no detectable similarity to the ALA synthase sequences which have been characterized from a variety of organisms, but are very similar to the cloned Salmonella typhimurium hemA sequences (T. Elliott, personal communication). Results of S1 nuclease protection experiments showed that the hemA mRNA appeared to have two different 5' ends and that a nonoverlapping divergent transcript was present upstream of the putative distal hemA transcriptional start site.

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.

Isolation and nucleotide sequence of the hemA gene of Escherichia coli K12.

The hemA gene of Escherichia coli K12 was cloned by complementation of a hemA mutant of this organism. Subcloning of the initial 6.0 kb HindIII fragment allowed the isolation of a 1.5 kb NheI-AvaI fragment which retained the ability to complement the hemA mutant. DNA sequencing by the dideoxy chain terminator method of Sanger showed the presence of an open reading frame (ORF) of 1254 nucleotides, which ends 6 nucleotides beyond the AvaI site. Primer extension experiments showed the existence of a putative transcription initiation site for the hemA gene, located at position 130. A possible promoter sequence was identified upstream from this transcription initiation site, and its functional activity was confirmed by the use of the pK01 promoter-probe vector. Protein synthesis in an in vitro coupled transcription-translation system showed a 46 kDa protein, which corresponds to the mol. wt. of the hemA protein, as deduced from the nucleotide sequence of the gene. No homology was found between the amino acid sequence of the hemA protein of E. coli K12 and known sequences of other delta-aminolevulinic acid synthases (delta-ALAS), suggesting that this protein is different from other delta-ALAS enzymes.

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

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.

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

The hemA gene which encodes δ-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 Ω 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.

Cloning and characterization of the 5-aminolevulinate synthase gene(s) from Rhodobacter sphaeroides

The 5-aminolevulinate synthase gene ( hemA) from Rhizobium meliloti was used to probe a genomic λ bank derived from Rhodobacter sphaeroides DNA. Two phage clones were found to bear homology to the Rhizobium probe. Southern hybridization analysis of the two λ phage clones, which we designated λHem10 and λHem12, showed that the homology to the Rhizobium hemA gene was localized to a 3.1-kb SalI fragment derived from λHem10 and a 7.0-kb SalI fragment derived from λHem12. Each of the SalI fragments was subsequently cloned into the multiple cloning site of pUC19 in both orientations relative to the lac promoter. Restriction analysis confirmed that each SalI fragment was unique. It was also shown from Southern hybridization analysis that the regions of homology within each of the R. sphaeroides restriction fragments and the Rhizobium probe were different. Further, we have tentatively concluded that each R. sphaeroides hemA gene shows a relatively low degree of homology to the other. Data obtained from in vitro transcription-translation studies in a homologous R. sphaeroides cell-free system, and complementation of hemA mutations of both Escherichia coli and R. sphaeroides by either of the putative hemA clones suggested the presence of a gene encoding 5-aminolevulinate synthase on each DNA sequence. The fact that 5-aminolevulinate synthase activity could be demonstrated in mutant strains complemented in trans with either cloned DNA fragment further supported this conclusion.