Mutant Strains of Rhodopseudomonas spheroides Lacking δ-Aminolevulinate Synthase: Growth, Heme, and Bacteriochlorophyll Synthesis

Two mutant strains of Rhodopseudomonas spheroides were described which lacked delta-aminolevulinate synthase activity. They required delta-aminolevulinate for growth; they did not respond to protoporphyrin or magnesium photoporphyrin, and only poorly to hemin. Synthesis of cytochromes and heme by mutant H-4 was dependent upon delta-aminolevulinate; this strain did not form bacteriochlorophyll either with or without delta-aminolevulinate and, consequently, grew only under aerobic conditions. Mutant H-5 formed bacteriochlorophyll in response to delta-aminolevulinate and grew both anaerobically in the light and aerobically in the dark; the amount of delta-aminolevulinate needed for optimal anaerobic growth was higher than that required aerobically. Synthesis of bacteriochlorophyll and heme by suspensions of mutant H-5 incubated anaerobically in the light was dependent upon delta-aminolevulinate; bacteriochlorophyll production was completely inhibited by high aeration and by puromycin. The mutants differed in their ability to take up radioactive delta-aminolevulinate from the external environment; mutant H-5 was less active than mutant H-4 or the wild type. It was suggested that R. spheroides made only one form of delta-aminolevulinate synthase, which provided delta-aminolevulinate for bacteriochlorophyll and heme synthesis.     

Nitrate,fumarate, and oxygen as electron acceptors for a late step in microbial heme synthesis

Nitrate can serve as anaerobic electron acceptor for the oxidation of protoporphyrinogen to protoporphyrin in cell-free extracts of Escherichia coli grown anaerobically in the presence of nitrate. Two kinds of experiments indicated this: anaerobic protoporphyrin formation from protoporphyrinogen, followed spectrophotometrically, was markedly stimulated by addition of nitrate; and anaerobic protoheme formation from protoporphyrinogen, determined by extraction procedures, was markedly stimulated by addition of nitrate. In contrast, anaerobic protoheme formation from protoporphyrin was not dependent upon addition of nitrate. This was the first demonstration of the ability of nitrate to serve as electron acceptor for this late step of heme synthesis. Previous studies with mammalian and yeast mitochondria had indicated an obligatory requirement for molecular oxygen at this step.In confirmation of our previous preliminary report, fumarate was also shown to be an electron acceptor for anaerobic protoporphyrinogen oxidation in extracts of E. coli grown anaerobically on fumarate. For the first time, anaerobic protoheme formation from protoporphyrinogen, but not from protoporphyrin, was shown to be dependent upon the addition of fumarate.The importance of these findings is 2-fold. First, they establish that enzymatic protoporphyrinogen oxidation can occur in the absence of molecular oxygen, in contrast to previous observations using mammalian and yeast mitochondria. Secondly, these findings help explain the ability of some facultative and anaerobic bacteria to form very large amounts of heme compounds, such as cytochrome pigments, when grown anaerobically in the presence of nitrate or fumarate. In fact, denitrifying bacteria are known to form more cytochromes when grown anaerobically than during aerobic growth.An unexpected finding was that extracts of another bacterium, Staphylococcus epidermidis, exhibited very little ability to oxidize protoporphyrinogen to protoporphyrin as compared to E. coli extracts. This finding suggests some fundamental differences in these two organisms in this key step in heme synthesis. It is known that these two facultative organisms also differ in that E. coli synthesizes cytochrome during both aerobic and anaerobic growth, while Staphylococcus only synthesizes cytochromes when grown aerobically.     

Inhibitory activity of Streptococcus mitis against oral bacteria

The antagonistic properties of three strains of Streptococcus mitis were investigated. They were found to inhibit a wide range of oral bacteria; Gram-positive and Gram-negative, facultative and anaerobic species being susceptible. The S. mitis strains were shown to be producing hydrogen peroxide, this being partially responsible for the aerobic inhibitory activity. A second inhibitory factor(s) was also produced, aerobically and anaerobically, although this could not be isolated. A limited characterization of this factor was undertaken using plate cultures.     

Specificity of the heme requirement for growth of Bacteroides ruminicola

Caldwell, D. R. (U.S. Department of Agriculture, Beltsville, Md.), D. C. White, M. P. Bryant, and R. N. Doetsch. Specificity of the heme requirement for growth of Bacteroides ruminicola. J. Bacteriol. 90:1645-1654. 1965.-Previous studies suggested that most strains of Bacteroides ruminicola subsp. ruminicola require heme for growth. Present studies with heme-requiring strain 23 showed that protoheme was replaced by various porphyrins, uroporphyrinogen, coproporphyrinogen, certain iron-free metalloporphyrins, hemes, and certain heme-proteins containing readily removable hemes. Strain 23 utilized a wider range of tetrapyrroles than hemin-requiring bacteria previously studied. Inactive compounds included porphyrin biosynthesis intermediates preceding the tetrapyrrole stage and related compounds; uroporphyrin, chlorophyll, pheophytin, phycoerythrin, bilirubin, pyrrole, FeSO(4) with or without chelating agents; and representative ferrichrome compounds. Strain 23, two other strains representing predominant biotypes of B. ruminicola subsp. ruminicola, and one closely related strain grew in media containing heme-free protoporphyrin, mesoporphyrin, hematoporphyrin, or deuteroporphyrin, apparently inserting iron into several nonvinyl porphyrins. Porphobilinogen and porphyrin synthesis, apparently via the commonly known heme synthesis pathway, occurred during growth of heme-independent B. ruminicola subsp. brevis strain GA33 in a tetrapyrrole-free medium containing delta-aminolevulinic acid, but delta-aminolevulinic acid metabolism to porphobilinogen or porphyrins could not be detected in cells of heme-requiring strain 23 grown in the same medium with hemin added. Growth of strain 23 with uroporphyrinogen, coproporphyrinogen, or protoporphyrin IX replacing hemin suggests that part of the commonly known heme-biosynthesis pathway is present in this strain, but nutritional and metabolic evidence indicates that some or all of the enzymes synthesizing the tetrapyrrole nucleus from linear molecules are lacking or inactive.     

Role of PUG1 in inducible porphyrin and heme transport in Saccharomyces cerevisiae

Unlike pathogenic fungi, the budding yeast Saccharomyces cerevisiae is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of PUG1 (protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing PUG1 exhibited decreased accumulation of [(55)Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of PUG1 overexpression on intracellular heme pools, we used a CYC1-lacZ reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.     

Microbial oxidation of protoporhydrinogen, an intermediate in heme and chlorphyll biosynthesis.

The oxidation of protoporphyrinogen to protoporphyrin, a late step in heme and chlorophyll synthesis, is catalyzed aerobically by a particulate fraction of Escherichia coli at a rate significantly higher than the rate of autooxidation. This activity is heat labile and is markedly inhibited by addition of respiratory substrates such as NADH. NADH is oxidized at a rate 100-fold higher than protoporphyrinogen. Particles from a cytochrome-less mutant of E. coli were markedly deficient in protoporphyrinogen oxidizing activity. Particles from a quinone-deficient mutant were also deficient. These findings suggest a possible role for the electron transport system in aerobic protoporphyrinogen oxidation. This activity was also examined in a variety of other bacteria. Particles from Streptococcus faecalis, which does not synthesize heme, were unable to oxidize protoporphyrinogen, confirming the specificity of this activity. Particles from aerobically grown Staphylococcus aureus exhibited protoporphyrinogen oxidizing activity, but particles from anaerobically grown cells had no activity above that of the nonenzymatic control. This indicates the repressible nature of this activity, and may also explain why Staphylococci synthesize cytochromes during aerobic, but not during anaerobic growth. Particles from photosynthetically grown Rhodopseudomonas spheroides, which contain both chlorophyll and heme, oxidized protoporphyrinogen at a rate no higher than the nonenzymic control. However, particles from cells grown aerobically, when bacteriochlorophyll synthesis is markedly repressed, readily exhibited protoporphyrinogen oxidizing activity. These initial findings suggest that this activity is detectable in cells primarily synthesizing heme, but not in cells primarily synthesizing bacteriochlorophyll, and could have implications both for the mechanism and regulation of the heme and bacteriochlorophyll pathways.     

Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene.

A Tn5-induced mutant of Bradyrhizobium japonicum, strain LORBF1, was isolated on the basis of the formation of fluorescent colonies, and stable derivatives were constructed in backgrounds of strains LO and I110. The stable mutant strains LOek4 and I110ek4 were strictly dependent upon the addition of exogenous hemin for growth in liquid culture and formed fluorescent colonies. The fluorescent compound was identified as protoporphyrin IX, the immediate precursor of protoheme. Cell extracts of strains LOek4 and I110ek4 were deficient in ferrochelatase activity, the enzyme which catalyzes the incorporation of ferrous iron into protoporphyrin IX to produce protoheme. Mutant strain I110ek4 could take up 55Fe from the growth medium, but, unlike the parent strain, no significant incorporation of radiolabel into heme was found. This observation shows that heme was not synthesized in mutant strain I110ek4 and that the heme found in those cells was derived from exogenous hemin in the growth medium. The putative protein encoded by the gene disrupted in strain LORBF1 and its derivatives was homologous to ferrochelatases from eukaryotic organisms. This homology, along with the described mutant phenotype, provides strong evidence that the disrupted gene is hemH, that which encodes ferrochelatase. Mutant strain I110ek4 incited nodules on soybean that did not fix nitrogen, contained few viable bacteria, and did not express leghemoglobin heme or apoprotein. The data show that B. japonicum ferrochelatase is essential for normal nodule development.     

Ferric-salicylaldehyde isonicotinoyl hydrazone, a synthetic iron chelate, alleviates defective iron utilization by reticulocytes of the Belgrade rat

The Belgrade rat has a hypochromic, microcytic anemia inherited as an autosomal recessive mutation. Although transferrin binds normally to reticulocytes and internalizes normally, iron accumulation into cells and heme is much slower than normal. We have investigated the role of the transferrin cycle in this mutant by bypassing transferrin iron delivery with the iron chelate ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH). Fe-SIH increases iron uptake into heme by Belgrade reticulocytes, restoring it almost to normal levels. This increase indicates that Fe-SIH delivers iron to a step in iron utilization that is after the Belgrade defect. Depleting reticulocytes of transferrin did not alter these observations. Failure to achieve above normal rates of iron incorporation could indicate damage due to chronic intracellular iron deficiency. Also, iron delivery by Fe-SIH restored globin synthesis to near-normal levels in Belgrade reticulocytes. The rates of glycine incorporation into porphyrin and heme in Belgrade reticulocytes incubated with Fe2-transferrin or Fe-SIH paralleled the rates of iron incorporation into heme. These data are consistent with the concept that iron availability limits protoporphyrin formation in rat reticulocytes. The protoporphyrin used for heme synthesis is provided by de novo synthesis and not by a pool of pre-existing protoporphyrin. The Belgrade defect occurs in the movement of iron from transferrin to a step prior to the ferrous state and insertion into heme. This defect diminishes the synthesis of heme and, consequently, that of protoporphyrin and globin.     

Metal ion substrate inhibition of ferrochelatase

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k(cat) and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) and can be alleviated by the addition of metal complexing agents such as beta-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.     

Characterization of the Late Steps of Microbial Heme Synthesis: Conversion of Coproporphyrinogen to Protoporphyrin

Cell-free extracts of various cytochrome-containing, heterotrophic microorganisms were examined for ability to convert coproporphyrinogen to protoporphyrin. Extracts of Escherichia coli and Pseudomonas denitrificans readily accumulated large amounts of protoporphyrin when assayed under aerobic conditions. However, protoporphyrin did not accumulate under either aerobic or anaerobic conditions of assay or in the presence of various supplements in extracts of the aerobe Micrococcus lysodeikticus, the facultative anaerobe Staphylococcus aureus, or the anaerobe Vibrio succinogenes. Protoporphyrin also accumulated when extracts of E. coli and P. denitrificans were incubated aerobically with the early heme precursor, delta-amino levulinic acid (ALA). This protoporphyrin accumulation was markedly stimulated by the iron chelator, o-phenanthroline. Extracts of S. aureus and M. lysodeikticus accumulated coproporphyrin, but not protoporphyrin when incubated with ALA. The enzyme system in extracts of E. coli which converts coproporphyrinogen to protoporphyrin under aerobic conditions of assay was also partially characterized. This conversion was stimulated by the iron chelator, o-phenanthroline, the respiratory inhibitor, cyanide, and the reducing agent, thioglycolate. Dialysis of the extract did not diminish enzyme activity. Certain alternate electron acceptors and nitrite caused a marked inhibition of the conversion. These results indicate that this late step in heme synthesis, the conversion of coproporphyrinogen to protoporphyrin, can be readily demonstrated in extracts of some, but not all, cytochrome-containing bacteria and that the aerobic conversion in E. coli exhibits many characteristics similar to those demonstrated for the aerobic conversion previously studied in liver mitochondria.     

Effects of iron deficiency on heme biosynthesis in Rhizobium japonicum.

The effects of iron deficiency on heme biosynthesis in Rhizobium japonicum were examined. Iron-deficient cells had a decreased maximum cell yield and a decreased cytochrome content and excreted protoporphyrin into the growth medium. The activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (EC 2.3.1.37) and delta-aminolevulinic acid dehydrase (EC 4.2.1.24), were diminished in iron-deficient cells, but were returned to normal levels upon addition of iron to the cultures. The addition of iron salts, iron chelators, hemin, or protoporphyrin to cell-free extracts did not affect the activity of these enzymes. The addition of levulinic acid to iron-deficient cultures blocked protoporphyrin excretion and also resulted in high delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase activities. These results suggest the possibility that rhizobial heme biosynthesis in the legume root nodule may be affected by the release of iron from the host plant to the bacteroids.     

The theft of host heme by Gram-positive pathogenic bacteria

The element iron is essential for bacteria and plays a key role in the virulence and pathology of bacterial diseases. The largest reservoir of iron within the human body is in protoporphyrin IX, the compound commonly referred to as heme and bound by hemoglobin. For many years, the study of heme uptake in bacteria was restricted to Gram-negative organisms. However, recent studies have shed light on how bacteria containing a thick peptidoglycan, such as Gram-positive bacteria, acquire and transport heme. This review summarizes old and new research covering the acquisition, transport, and utilization of heme in Gram-positive bacterial pathogens.     

Regulation of rat hepatic delta-aminolevulinic acid synthetase and heme oxygenase activities: evidence for control by heme and against mediation by prosthetic iron

1. The effect of in vivo administration of 6 compounds on the activity of delta-aminolevulinic acid (ALA) synthetase and heme oxygenase were determined. 2. The order of decreasing potency in reducing ALA synthetase activity was heme, bilirubin, protoporphyrin IX, bilirubin dimethyl ester, CoCl2 and FeCl3. 3. The chelating agents EDTA and deferoxamine did not prevent heme's repression of ALA synthetase or induction of heme oxygenase activity. 4. The dose response, time course, enzyme subcellular distribution and chelation antagonism studies all suggest that heme itself, and not iron, regulates the rate limiting enzymatic steps of rat hepatic heme synthesis and degradation.     

Hemolytic activity reevaluation of putative nonpathogenic Listeria monocytogenes strains.

Identification of 12 strains originally characterized as nonpathogenic Listeria monocytogenes was reassured following the evaluation of their hemolytic capability with a newly developed horse blood agar plate. Seven of the strains were observed consistently to be hemolytic and confirmed as L. monocytogenes with the use of two commercial systems: the Gene-Trak L. monocytogenes-specific colorimetric DNA hybridization assay and the API Listeria system. Except for one strain that formed typical smooth colonies, these hemolytic strains formed rough colonies on a selective medium, lithium chloride-ceftazidime agar. The rest of the strains were nonhemolytic and did not hybridize with the DNA probe; they were identified as Listeria innocua on the basis of their API Listeria system biochemical profile. All but one of these nonhemolytic strains formed smooth colonies on lithium chloride-ceftazidime agar.     

Identification of a secreted cholesterol-dependent cytolysin (mitilysin) from Streptococcus mitis

We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of Streptococcus mitis isolates. We have sequenced the mitilysin gene from seven isolates of S. mitis. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions. S. mitis appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of S. mitis may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between S. mitis and Streptococcus pneumoniae, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.     

X-ray crystallographic and biochemical characterization of the inhibitory action of an imidazole-dioxolane compound on heme oxygenase

Heme oxygenase (HO) catalyzes the regiospecific cleavage of the porphyrin ring of heme using reducing equivalents and O2 to produce biliverdin, iron, and CO. Because CO has a cytoprotective effect through the p38-MAPK pathway, HO is a potential therapeutic target in cancer. In fact, inhibition of the HO isoform HO-1 reduces Kaposi sarcoma tumor growth. Imidazole-dioxolane compounds have recently attracted attention because they have been reported to specifically inhibit HO-1, but not HO-2, unlike Cr-containing protoporphyrin IX, a classical inhibitor of HO, that inhibits not only both HO isoforms but also other hemoproteins. The inhibitory mechanism of imidazole-dioxolane compounds, however, has not yet been characterized. Here, we determine the crystal structure of the ternary complex of rat HO-1, heme, and an imidazole-dioxolane compound, 2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane. This compound bound on the distal side of the heme iron, where the imidazole and 4-chlorophenyl groups were bound to the heme iron and the hydrophobic cavity in HO, respectively. Binding of the bulky inhibitor in the narrow distal pocket shifted the distal helix to open the distal site and moved both the heme and the proximal helix. Furthermore, the biochemical characterization revealed that the catalytic reactions of both HO-1 and HO-2 were completely stopped after the formation of verdoheme in the presence of the imidazole-dioxolane compound. This result should be mainly due to the lower reactivity of the inhibitor-bound verdoheme with O2 compared to the reactivity of the inhibitor-bound heme with O2.     

Uric Acid-Degrading Bacteria in Guts of Termites [Reticulitermes flavipes (Kollar)

Uricolytic bacteria were present in guts of Reticulitermes flavipes in populations up to 6 x 10 cells per gut. Of 82 strains isolated under strict anaerobic conditions, most were group N Streptococcus sp., Bacteroides termitidis, and Citrobacter sp. All isolates used uric acid (UA) as an energy source anaerobically, but not aerobically, and NH(3) was the major nitrogenous product of uricolysis. However, none of the isolates had an absolute requirement for UA. Utilization of heterocyclic compounds other than UA was limited. Fresh termite gut contents also degraded UA anaerobically, as measured by CO(2) evolution from [2-C]UA. The magnitude of anaerobic uricolysis [0.67 pmol of UA catabolized/(gut x h)] was entirely consistent with the population density of uricolytic bacteria in situ. Uricolytic gut bacteria may convert UA in situ to products usable by termites for carbon, nitrogen, energy, or all three. This possibility is consistent with the fact that R. flavipes termites from UA, but they do not void the purine in excreta despite the lack of uricase in their tissues.     

Interaction between the bacterial iron response regulator and ferrochelatase mediates genetic control of heme biosynthesis.

The heme biosynthetic pathway culminates with the insertion of iron into protoporphyrin catalyzed by ferrochelatase. The Bradyrhizobium japonicum iron response regulator (Irr) protein represses the pathway at an early step under iron limitation to prevent protoporphyrin synthesis from exceeding iron availability. Here, we show that Irr interacts directly with ferrochelatase and responds to iron via the status of heme and protoporphyrin localized at the site of heme synthesis. In the presence of iron, ferrochelatase inactivates Irr, followed by heme-dependent Irr degradation to derepress the pathway. Under iron limitation, protoporphyrin relieves the inhibition of Irr by ferrochelatase, probably by promoting protein dissociation, allowing genetic repression. Thus, metabolic control of the heme pathway involves a regulatory function of a biosynthetic enzyme to affect gene expression. Furthermore, heme can serve as a signaling molecule without accumulating freely in cells.     

Interaction of heme and heme-hemopexin with an extracellular oxidant system used to measure cell growth-associated plasma membrane electron transport

Since redox active metals are often transported across membranes into cells in the reduced state, we have investigated whether exogenous ferri-heme or heme bound to hemopexin (HPX), which delivers heme to cells via receptor-mediated endocytosis, interact with a cell growth-associated plasma membrane electron transport (PMET) pathway. PMET reduces the cell-impermeable tetrazolium salt, WST-1, in the presence of the mandatory low potential intermediate electron acceptor, mPMS. In human promyelocytic (HL60) cells, protoheme (iron protoporphyrin IX; 2,4-vinyl), mesoheme (2,4-ethyl) and deuteroheme (2,4-H) inhibited reduction of WST-1/mPMS in a saturable manner supporting interaction with a finite number of high affinity acceptor sites (Kd 221 nM for naturally occurring protoheme). A requirement for the redox-active iron was shown using gallium-protoporphyrin IX (PPIX) and tin-PPIX. Heme-hemopexin, but not apo-hemopexin, also inhibited WST-1 reduction, and copper was required. Importantly, since neither heme nor heme-hemopexin replace mPMS as an intermediate electron acceptor and since inhibition of WST-1/mPMS reduction requires living cells, the experimental evidence supports the view that heme and heme-hemopexin interact with electrons from PMET. We therefore propose that heme and heme-hemopexin are natural substrates for this growth-associated electron transfer across the plasma membrane.