Expression Of hemA Research Articles

Escherichia coli with increased aminoglycoside resistance due to an amino acid substitution at position 85 of HemC

ObjectiveThe mechanism of aminoglycoside resistance due to abnormal hemin synthesis remains unclear. We investigate an Escherichia coli strain with a single amino acid substitution at position 85 of HemC. MethodsAn aminoglycoside-resistant Escherichia coli DH5α was selected by passaging in Lysogeny Broth (LB) medium containing amikacin. Whole genome sequencing was performed to determine the genetic profile of the strain. An isogenic strain of E. coli DH5α was created. Growth rates, drug susceptibilities and expressions of the heme synthetic genes were compared between the original strain and the isogenic strain. ResultsWhole genome sequencing revealed a nucleotide substitution at position 254 of hemC from adenine (A) to thymine (T), resulting in an amino acid substitution at position 85 of HemC from histidine (H) to leucine (L). There were no mutations in other heme synthetic genes, including hemA, hemB, hemC, hemD, hemE, hemF, hemG, hemH, hemL, hemN, hemX and hemY. The isogenic strain of E. coli DH5α with H85L in HemC was less susceptible to aminoglycosides, and its growth was slower than that of E. coli DH5α before passage. Quantitative real-time PCR showed that the expression of hemA was higher and the expressions of hemL, hemG and hemX lower in the isogenic strain than before passage. ConclusionThis is the first report of aminoglycoside resistance due to an amino acid substitution in HemC. These findings suggested that mutations in the heme synthetic genes may indirectly affect the growth rates of E. coli strains and their susceptibilities to aminoglycosides.

HemU and TonB1 contribute to hemin acquisition in Stenotrophomonas maltophilia.

The hemin acquisition system is composed of an outer membrane TonB-dependent transporter that internalizes hemin into the periplasm, periplasmic hemin-binding proteins to shuttle hemin, an inner membrane transporter that transports hemin into the cytoplasm, and cytoplasmic heme oxygenase to release iron. Fur and HemP are two known regulators involved in the regulation of hemin acquisition. The hemin acquisition system of Stenotrophomonas maltophilia is poorly understood, with the exception of HemA as a TonB-dependent transporter for hemin uptake. Putative candidates responsible for hemin acquisition were selected via a homolog search and a whole-genome survey of S. maltophilia. Operon verification was performed by reverse transcription-polymerase chain reaction. The involvement of candidate genes in hemin acquisition was assessed using an in-frame deletion mutant construct and iron utilization assays. The transcript levels of candidate genes were determined using quantitative polymerase chain reaction. Smlt3896-hemU-exbB2-exbD2-tonB2 and tonB1-exbB1-exbD1a-exbD1b operons were selected as candidates for hemin acquisition. Compared with the parental strain, hemU and tonB1 mutants displayed a defect in their ability to use hemin as the sole iron source for growth. However, hemin utilization by the Smlt3896 and tonB2 mutants was comparable to that of the parental strain. HemA expression was repressed by Fur in iron-replete conditions and derepressed in iron-depleted conditions. HemP negatively regulated hemA expression. Like hemA, hemU was repressed by Fur in iron-replete conditions; however, hemU was moderately derepressed in response to iron-depleted stress and fully derepressed when hemin was present. Unlike hemA and hemU, the TonB1-exbB1-exbD1a-exbD1b operon was constitutively expressed, regardless of the iron level or the presence of hemin, and Fur and HemP had no influence on its expression. HemA, HemU, and TonB1 contribute to hemin acquisition in S. maltophilia. Fur represses the expression of hemA and hemU in iron-replete conditions. HemA expression is regulated by low iron levels, and HemP acts as a negative regulator of this regulatory circuit. HemU expression is regulated by low iron and hemin levels in a hemP-dependent manner.

Function of ALA Content in Porphyrin Metabolism Regulation of Ananas comosus var. bracteatus.

Chlorophyll and heme are essential molecules for photosynthesis and respiration, which are competing branches of the porphyrin metabolism pathway. Chlorophyll and heme balance regulation is very important for the growth and development of plants. The chimeric leaves of Ananas comosus var. bracteatus were composed of central photosynthetic tissue (PT) and marginal albino tissue (AT), which were ideal materials for the study of porphyrin metabolism mechanisms. In this study, the regulatory function of ALA content on porphyrin metabolism (chlorophyll and heme balance) was revealed by comparing PT and AT, 5-Aminolevulinic Acid (ALA) exogenous supply, and interference of hemA expression. The AT remained similar in porphyrin metabolism flow level to the PT by keeping an equal ALA content in both tissues, which was very important for the normal growth of the chimeric leaves. As the chlorophyll biosynthesis in AT was significantly inhibited, the porphyrin metabolism flow was directed more toward the heme branch. Both tissues had similar Mg2+ contents; however, Fe2+ content was significantly increased in the AT. The chlorophyll biosynthesis inhibition in the white tissue was not due to a lack of Mg2+ and ALA. A 1.5-fold increase in ALA content inhibited chlorophyll biosynthesis while promoting heme biosynthesis and hemA expression. The doubling of ALA content boosted chlorophyll biosynthesis while decreasing hemA expression and heme content. HemA expression interference resulted in a higher ALA content and a lower chlorophyll content, while the heme content remained at a relatively low and stable level. Conclusively, a certain amount of ALA was important for the stability of porphyrin metabolism and the normal growth of plants. The ALA content appears to be able to regulate chlorophyll and heme content by bidirectionally regulating porphyrin metabolism branch direction.

Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli

Strain engineering and bioprocessing strategies were applied for biobased production of porphobilinogen (PBG) using Escherichia coli as the cell factory. The non-native Shemin/C4 pathway was first implemented by heterologous expression of hemA from Rhodopseudomonas spheroids to supply carbon flux from the natural tricarboxylic acid (TCA) pathways for PBG biosynthesis via succinyl-CoA. Metabolic strategies were then applied for carbon flux direction from the TCA pathways to the C4 pathway. To promote PBG stability and accumulation, Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) was applied to repress hemC expression and, therefore, reduce carbon flowthrough toward porphyrin biosynthesis with minimal impact to cell physiology. To further enhance PBG biosynthesis and accumulation under the hemC-repressed genetic background, we further heterologously expressed native E. coli hemB. Using these engineered E. coli strains for bioreactor cultivation based on ~ 30 g L−1 glycerol, we achieved high PBG titers up to 209 mg L−1, representing 1.73% of the theoretical PBG yield, with improved PBG stability and accumulation. Potential biochemical, genetic, and metabolic factors limiting PBG production were systematically identified for characterization.Graphical

Transcriptomic analysis for elucidating the physiological effects of 5-aminolevulinic acid accumulation on Corynebacterium glutamicum

5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthetic pathway, attracts close attention among researchers because of its potential applications to cancer treatment and agriculture. Overexpression of heterologous hemA and hemL, which encode glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase, respectively, in Corynebacterium glutamicum produces ALA, although whether ALA accumulation causes unintended effects on the host is unknown. Here we used an integrated systems approach to compare global transcriptional changes induced by the expression of hemA and hemL. Metabolic pathway such as glycolysis was inhibited, but tricarboxylic acid cycle, pentose phosphate pathway, and respiratory metabolism were stimulated. Moreover, the transcriptional levels of certain genes involved in heme biosynthesis were up-regulated, and the data implicate the two-component system (TCS) HrrSA was involved in the regulation of heme synthesis. With these understandings, it is proposed that ALA accumulation stimulates heme synthesis pathway and respiratory metabolism. Our study illuminates the physiological effects of overexpressing hemA and hemL on the phenotype of C. glutamicum and contributes important insights into the regulatory mechanisms of the heme biosynthetic pathways.

Influence of exogenous 5-aminolevulinic acid on chlorophyll synthesis and related gene expression in oilseed rape de-etiolated cotyledons under water-deficit stress

5-aminolevulinic acid (ALA) is an essential precursor for the biosynthesis of tetrapyrrols such as heme and chlorophyll (Chl). Previous studies have focused mainly on promotive effects of exogenous ALA on plant growth, while regulatory mechanisms affecting Chl biosynthesis have been only partially discussed. In the present study, the ameliorative role of exogenous ALA was investigated on Chl and endogenous ALA biosynthesis in six-day-old etiolated oilseed rape (Brassica napus L.) cotyledons during the de-etiolation stage. We showed that exogenously applied ALA of a low dosage enhanced Chl and ALA accumulation in cotyledons, while 600 µM ALA treatment inhibited the accumulation of Chl and ALA severely. However, the gene expression levels of glutamyl-tRNA reductase (HEMA) and glutamate-1-semialdehyde aminotransferase (GSA) were not affected under either low or high ALA concentrations. Furthermore, water deficit induced by polyethylene glycol 6000 (PEG) suppressed the Chl and ALA accumulation in cotyledons, while the inhibition was partially alleviated in the cotyledons pretreated with ALA. The decrease in Chl biosynthesis induced by PEG stress was assumed to be related to downregulation of HEMA and Mg-chelatase ChlH (ChlH), and upregulation of ferrochelatase (FC) genes. Moreover, exogenously applied ALA did not show any effect on the expression of Chl synthesis-related genes under the PEG treatment. These results showed a difference in suppressing ALA synthesis due to the high concentration of ALA and PEG. Exogenously applied ALA did not affect the expression of HEMA and GSA, thus exogenous ALA regulated Chl synthesis not via the regulation of transcriptional level in ALA biosynthesis. However, the inhibition in Chl and endogenous ALA accumulation by the PEG treatment may be attributed to downregulation of HEMA and ChlH, and upregulation of FC.

The regulation of cytotoxicity and cyclooxygenase‐2 expression by 2‐hydroxy‐ethyl methacrylate in human osteoblasts are related to intracellular glutathione levels

To investigate the effects of 2-hydroxy-ethyl methacrylate (HEMA) on cytotoxicity and cyclooxygenase-2 (COX-2) protein expression in human osteoblasts. Cytotoxicity was judged using an Alamar Blue reduction assay on human osteoblast cell line U2OS. Western blot was used to evaluate the expression of COX-2 protein by HEMA. To determine whether glutathione (GSH) levels were important in cytotoxicity and COX-2 expression of HEMA, cells were pre-treated with the GSH precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. Paired Student's t-tests were applied for the statistical analysis of the results. HEMA demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P<0.05). The 50% inhibition concentration of HEMA was approximately 3mmol L(-1) . HEMA was found to induce COX-2 protein expression in U2OS cells (P<0.05). The addition of OTZ acted as a protective effect on HEMA-induced cytotoxicity and COX-2 expression (P<0.05). In contrast, the addition of BSO enhanced HEMA-induced cytotoxicity and COX-2 expression (P<0.05). Taken together, the levels of HEMA that were tested inhibited cell growth on U2OS cells. HEMA has a significant potential for periapical toxicity. The activation of COX-2 protein expression may be one of the mechanisms of HEMA-induced periapical inflammation. These inhibitory effects were associated with intracellular GSH levels.

Analysis of the role of theAspergillus nigeraminolevulinic acid synthase (hemA) gene illustrates the difference between regulation of yeast and fungal haem- and sirohaem-dependent pathways

To increase knowledge on haem biosynthesis in filamentous fungi like Aspergillus niger, pathway-specific gene expression in response to haem and haem intermediates was analysed. This analysis showed that iron, 5'-aminolevulinic acid (ALA) and possibly haem control haem biosynthesis mostly via modulating expression of hemA [coding for 5'-aminolevulinic acid synthase (ALAS)]. A hemA deletion mutant (ΔhemA) was constructed, which showed conditional lethality. Growth of ΔhemA was supported on standard nitrate-containing media with ALA, but not by hemin. Growth of ΔhemA could be sustained in the presence of hemin in combination with ammonium instead of nitrate as N-source. Our results suggest that a branch-off within the haem biosynthesis pathway required for sirohaem synthesis is responsible for lack of growth of ΔhemA in media containing nitrate as sole N-source, because of the requirement of sirohaem for nitrate assimilation, as a cofactor of nitrite reductase. In contrast to the situation in Saccharomyces cerevisiae, cysteine, but not methionine, was found to further improve growth of ΔhemA. These results demonstrate that A. niger can use exogenous hemin for its cellular processes. They also illustrate important differences in regulation of haem biosynthesis and in the role of haem and sirohaem in A. niger compared to S. cerevisiae.

Regulation of hem Gene Expression in Rhodobacter capsulatus by Redox and Photosystem Regulators RegA, CrtJ, FnrL, and AerR

Biosynthetic pathways for heme and chlorophyll share common intermediates from 5-aminolevulinic acid through protoporphyrin IX. To obtain a better understanding of how photosynthetic organisms coordinate heme and chlorophyll biosynthesis, we have undertaken detailed analysis of the expression pattern of numerous heme biosynthesis genes in the purple photosynthetic bacterium Rhodobacter capsulatus. beta-Galactosidase reporter assays demonstrated that expression of hemA, hemB, hemC, hemE and hemZ genes is elevated under conditions that give rise to elevated bacteriochlorophyll synthesis. Heme gene expression is shown to be affected by mutations in previously identified transcriptional regulators RegA, FnrL, CrtJ, and AerR, which also control expression of genes involved in bacteriochlorophyll and carotenoid synthesis, and synthesis of the apoprotein subunits of the photosynthetic and electron transport apparatus. High-resolution primer extension analysis of hem mRNA reveals the presence of numerous putative RegA, FnrL and CrtJ binding sites in several hem promoter regions.

Analysis of the upstream sequences of the Rhodobacter sphaeroides 2.4.1 hemA gene: in vivo evidence for the presence of two promoters that are both regulated by fnrL.

The presumed DNA target for the Rhodobacter sphaeroides 2.4.1 FnrL regulatory protein is the FNR consensus sequence, TTGAT-N(4)-ATCAA, based on (1) similarities between the helix-turn-helix motifs of FnrL and the Escherichia coli homologue, Fnr, and (2) the established FnrL dependence for anaerobic induction of six gene clusters all having upstream FNR consensus-like sequences. We are interested in understanding the regulation of one among these; namely, the hemA gene, which codes for one of two isozymes of 5-aminolevulinate (ALA) synthase in this organism. Here, we present in vivo evidence that the hemA gene is transcribed from two promoters. Both are under oxygen control, and disabling the fnrL gene abolishes induction of each promoter in response to lowering oxygen tension. Based on the 5' position of the FNR consensus sequence relative to the downstream promoter, we had hypothesized that activation of that promoter is mediated by binding of FnrL to the FNR consensus sequence. Consistent with this hypothesis, we found here that transcription from the downstream promoter is no longer inducible when the FNR consensus sequence is deleted. With respect to the upstream promoter, based on the fact that the +1 site of transcription from that promoter is within the FNR consensus sequence, we propose an indirect role for FnrL. This possibility is discussed, together with other unresolved aspects of hemA expression.

Expression of glutamyl-tRNA reductase in Escherichia coli

The biosynthesis of the hemes, chlorophylls, corrins and other tetrapyrroles begins with the synthesis of 5-aminolevulinic acid (ALA). The pathway is highly conserved except for the synthesis of ALA which is derived from glycine and succinyl CoA (C4) in most eukaryotes and from glutamate (C5) in most bacteria and in green plants. In C5, glutamyl-tRNA synthetase (GTS) converts glutamate to glutamyl-tRNA (glu-tRNA), which is reduced by glutamyl-tRNA reductase (GTR) to glutamyl-1-semialdehyde (GSA), which is converted by aminotransferase (GSA-AT) to ALA. Since GTS is also involved in protein synthesis and GSA can be converted to ALA non-enzymatically, it is highly probable that control of ALA synthesis and thus of the whole pathway resides in the GTR step. In Escherichia coli, GTR is the gene product of hemA. BL21(DE 3), a protease-deficient strain which contains the T7 RNA polymerase gene in front of a lac promoter, was transformed with a pET14b-based vector, pWC01, harboring hemA in front of a T7 promoter and ORF1 which is transcribed in the opposite direction. The transformed strain, WC1201, secreted ALA and porphyrins into the medium. Induction of expression of hemA by WC1201 was optimized for concentration of inducer (IPTG, 5 mM), temperature (37°C), presence of betaine and sorbitol (no change) and time of induction (2 h). GTR was observable as a 46 kDa band by Brilliant blue G staining of SDS-PAGE gels. Sonicates of the induction mixture exhibited strong ALA synthesis activity which was enhanced by tRNA glu. Most of the activity was in the supernatant of the sonicate indicating that GTR is a soluble enzyme. The induced strain had more GTS activity than the uninduced strain which had more GTS activity than its parent wild-type strain. Autoradiography on native gradient PAGE showed that GTR expressed in vivo by induction of WC1201 had a molecular weight of approx. 117 kDa. Gel filtration of the induced sonicate showed a peak of enzymatic activity at about 126 kDa. When pET14b- or pUC19-based plasmids harboring hemA and ORF1, or importantly, a pUC19-based plasmid harboring only hemA and not ORF1, were expressed in an in vitro transcription-translation system, native gradient PAGE showed a product with a molecular weight of approximately 175 kDA. This expression was higher in the presence of tRNA glu. When the 117 kDa and 175 kDa proteins were excised from their native gels respectively, and run on SDS PAGE, autoradiography showed bands at 46 kDa. We conclude that GTR is present in both high molecular weight species. Since overexpression of hemA from pET14b-based plasmids is associated with increased glutamyl-tRNA synthetase activity, the 175 kDa species may represent different complexes of GTR, GTS and glutamyl-tRNA as observed in Chlamydomonas and the 117–126 kDa species may be an dimer of GTR associated with glu-tRNA or a complex of GTR, GTS and glu-tRNA. These possibilities are being investigated.

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: regulation through alterations in the cellular redox state

Rhodobacter sphaeroides 2.4.1 has the ability to synthesize a variety of tetrapyrroles, reflecting the metabolic versatility of this organism and making it capable of aerobic, anaerobic, photosynthetic, and diazotrophic growth. The hemA and hemT genes encode isozymes that catalyze the formation of 5-aminolevulinic acid, the first step in the biosynthesis of all tetrapyrroles present in R. sphaeroides 2.4.1. As part of our studies of the regulation and expression of these genes, we developed a genetic selection that uses transposon mutagenesis to identify loci affecting the aerobic expression of the hemA gene. In developing this selection, we found that sequences constituting an open reading frame immediately upstream of hemA positively affect hemA transcription. Using a transposon-based selection for increased hemA expression in the absence of the upstream open reading frame, we isolated three independent mutants. We have determined that the transposon insertions in these strains map to three different loci located on chromosome 1. One of the transposition sites mapped in the vicinity of the recently identified R. sphaeroides 2.4.1 homolog of the anaerobic regulatory gene fnr. By marker rescue and DNA sequence analysis, we found that the transposition site was located between the first two genes of the cco operon in R. sphaeroides 2.4.1, which encodes a cytochrome c terminal oxidase. Examination of the phenotype of the mutant strain revealed that, in addition to increased aerobic expression of hemA, the transposition event also conferred an oxygen-insensitive development of the photosynthetic membranes. We propose that the insertion of the transposon in cells grown in the presence of high oxygen levels has led to the generation of a cellular redox state resembling either reduced oxygen or anaerobiosis, thereby resulting in increased expression of hemA, as well as the accumulation of spectral complex formation. Several models are presented to explain these findings.

Transcription of the glutamyl-tRNA reductase (hemA) gene in Salmonella typhimurium and Escherichia coli: role of the hemA P1 promoter and the arcA gene product.

In Salmonella typhimurium and Escherichia coli, the hemA gene encodes the enzyme glutamyl-tRNA reductase, which catalyzes the first committed step in the heme biosynthetic pathway. It has recently been reported that a lac operon fusion to the hemA promoter of E. coli is induced 20-fold after starvation for heme. Induction was dependent on the transcriptional regulator ArcA, with a second transcriptional regulator, FNR, playing a negative role specifically under anaerobic conditions (S. Darie and R. P. Gunsalus, J. Bacteriol. 176:5270-5276, 1994). We have investigated the generality of this effect by examining the response to heme starvation of a number of lac operon fusions to the hemA promoters of both E. coli and S. typhimurium. We confirmed that such fusions are induced during starvation of a hemA auxotroph, but the level of induction observed was maximally sixfold and for S. typhimurium fusions it was only two- to fourfold. Sequences required for high-level expression of hemA lie within 129 bp upstream of the major (P1) promoter transcriptional start site. Mutants defective in the P1 promoter had greatly reduced hemA-lac expression both in the presence and in the absence of ALA. Mutations in arcA had no effect on hemA-lac expression in E. coli during normal growth, although the increase in expression during starvation for ALA was half that seen in an arcA+ strain. Overexpression of the arcA gene had no effect on hemA-lac expression. Primer extension analysis showed that RNA 5' ends mapping to the hemA P1 and P2 promoters were not expressed at significantly higher levels in induced cultures. These results differ from those previously reported.

Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene.

In Rhodobacter sphaeroides 2.4.1, the cellular requirements for 5-aminolevulinic acid (ALA) are in part regulated by the level of ALA synthase activity, which is encoded by the hemA and hemT genes. Under standard growth conditions, only the hemA gene is transcribed, and the level of ALA synthase activity varies in response to oxygen tension. The presence of an FNR consensus sequence upstream of hemA suggested that oxygen regulation of hemA expression could be mediated, in part, through a homolog of the fnr gene. Two independent studies, one detailed here, identified a region of the R. sphaeroides 2.4.1 genome containing extensive homology to the fix region of the symbiotic nitrogen-fixing bacteria Rhizobium meliloti and Bradyrhizobium japonicum. Within this region that maps to 443 kbp on chromsome I, we have identified an fnr homolog (fnrL), as well as a gene that codes for an anaerobic coproporphyrinogen III oxidase, the second such gene identified in this organism. We also present an analysis of the role of fnrL in the physiology of R. sphaeroides 2.4.1 through the construction and characterization of fnrL-null strains. Our results further show that fnrL is essential for both photosynthetic and anaerobic-dark growth with dimethyl sulfoxide. Analysis of hemA expression, with hemA::lacZ transcriptional fusions, suggests that FnrL is an activator of hemA under anaerobic conditions. On the other hand, the open reading frame immediately upstream of hemA appears to be an activator of hemA transcription regardless of either the presence or the absence of oxygen or FnrL. Given the lack of hemT expression under these conditions, we consider FnrL regulation of hemA expression to be a major factor in bringing about changes in the level of ALA synthase activity in response to changes in oxygen tension.

5-Aminolevulinic acid synthesis in Escherichia coli requires expression of hemA.

hemA and hemM, which are 213 bp apart and divergently transcribed, were separately cloned. We found that hemA is required for 5-aminolevulinic acid (ALA) synthesis in two ALA- auxotrophs. Overexpression of hemM alone did not produce ALA. More ALA was produced by strains harboring a plasmid with both hemA and hemM than by those with hemA alone. We conclude that hemA alone is required for ALA synthesis but hemA and hemM are required for maximal ALA synthesis.