Response of arsenic-induced oxidative stress,DNA damage,and metal imbalance to combined administration of DMSA and monoisoamyl-DMSA during chronic arsenic poisoning in rats

Arsenic and its compounds cause adverse health effects in humans. Current treatment employs administration of thiol chelators, such as meso-2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercaptopropane 1-sulfonate (DMPS), which facilitate its excretion from the body. However, these chelating agents are compromised by number of limitations due to their lipophobic nature, particularly in case of chronic poisoning. Combination therapy is a new approach to ensure enhanced removal of metal from the body, reduced doses of potentially toxic chelators, and no redistribution of metal from one organ to another, following chronic metal exposure. The present study attempts to investigate dose-related effects of two thiol chelators, DMSA and one of its new analogues, monoisoamyl dimercaptosuccinic acid (MiADMSA), when administered in combination with the aim of achieving normalization of altered biochemical parameters suggestive of oxidative stress and depletion of inorganic arsenic following chronic arsenic exposure. Twenty-five adult male Wistar rats were given 25 ppm arsenic for 10 weeks followed by chelation therapy with the above chelating agents at a dose of 0.3 mmol/kg (orally) when administered individually or 0.15 mmol/kg and 0.3 mmol/kg (once daily for 5 consecutive days), respectively, when administered in combination. Arsenic exposure led to the inhibition of blood δ-aminolevulinic acid dehydratase (ALAD) activity and depletion of glutathione (GSH) level. These changes were accompanied by significant depletion of hemoglobin, RBC and Hct as well as blood superoxide dismutase (SOD) acitivity. There was an increase in hepatic and renal levels of thiobarbituric acid-reactive substances, while GSH:GSSG ratio decreased significantly, accompanied by a significant increase in metallothionein (MT) in hepatocytes. DNA damage based on denaturing polyacrylamide gel electrophoresis revealed significant loss in the integrity of DNA extracted from the liver of arsenic-exposed rats compared to that of normal animals. These changes were accompanied by a significant elevation in blood and soft-tissue arsenic concentration. Co-administration of DMSA and MiADMSA at lower dose (0.15 mmol/kg) was most effective not only in reducing arsenic-induced oxidative stress but also in depleting arsenic from blood and soft tissues compared to other treatments. This combination was also able to repair DNA damage caused following arsenic exposure. We thus recommend combined administration of DMSA and MiADMSA for achieving optimum effects of chelation therapy.     

Influence of zinc-saccharide complexes on some haematological parameters in rats

The effects of three recently synthesized zinc-saccharide complexes (zinc-fructose, zinc-galactose and zinc-glucose) on blood -aminolevulinic acid dehydratase (ALAD) activity, glutathione (GSH), zinc-protoporphyrin (ZPP) and urinary -aminolevulinic acid (ALA) levels have been investigated to ascertain the utility of these complexes as zinc supplements and as preventive agents against lead intoxication in rats.     

Capacity for chlorophyll synthesis in heat-bleached 70S ribosome-deficient rye leaves

The leaves of young rye plants (Secale cereale L.) grown at 32° were deficient in chlorophyll and in chloroplastic rRNA as compared to those grown at 22°, which developed normally. Both chlorophyll accumulation and the formation of plastidic rRNA were largely restored at 32° when the plants were transfered several times for 1 h per day to 22°. In the chlorotic 32°-grown rye leaves the in vivo activity of -aminolevulinate synthetase was very low. Aminolevulinate dehydratase however, exhibited high activity in extracts from 32°-grown leaves and was localized in the plastid fraction isolated from the chlorotic leaf tissue. After application of -aminolevulinic acid to chlorotic parts of leaves growing at 32°, protochlorophyll(ide) was formed and accumulated in the dark. In the light, the protochlorophyll(ide) was photooxidized at 32°. The results suggest a cytoplasmic site of synthesis for the series of enzymes converting -aminolevulinate to protochlorophyll(ide). It is concluded that an inhibition of -aminolevulinate synthetase and the photooxidation of protochlorophyll(ide) or chlorophyll are responsible for the chlorosis of the leaves at 32°.Abbreviations ALA -aminolevulinic acid - ALAD -aminolevulinate dehydratase - ALAS -aminolevulinate synthetase     

Comparisons of δ13C values in leaves of aquatic macrophytes from different habitats in Britain and Finland; some implications for photosynthetic processes in aquatic plants

Summary The ¹³C values of submerged aquatic plants from contrasting but relatively defined habitats, and the ¹³C values of emergent, floating and submerged leaves of dimorphic aquatic plants, were measured. In many instances the ¹³C values of dissolved inorganic carbon in the water were also measured. Plant ¹³C values in the vicinity of-40 to-50 were found in rapidly flowing spring waters with carbonate ¹³C values of-16 to-21, consistent with the notion that species such as Fontinalis antipyretica almost exclusively assimilate free CO₂ via RuP₂ carboxylase. Plant ¹³C values in the vicinity of-10 to-15 in sluggish water with carbonate ¹³C values of about-5 were observed, consistent with the notion that boundary layer diffusion and/or HCO₃ ^- uptake may determine the ¹³C value of submerged aquatic plants in these circumstances. Comparisons of ¹³C values of the same or related species growing in waters of similar carbonate ¹³C value but different flow rates confirmed this view; more negative ¹³C values were frequently associated with plants in fast moving water. In Britain, but not in Finland, the ¹³C values of submerged leaves of dimorphic plants were almost invariably more negative than in aerial leaves. The ¹³C value of carbonate from chalk streams and in acid springs indicate substantial inputs of respiratory CO₂, as opposed to atmospheric carbon. The contributions of these variations in ¹³C of the carbon source, and of isotope fractionation in diffusion, to the ¹³C value of submerged parts of dimorphic plants is discussed.     

Evidence for the interference of aluminum with bacterial porphyrin biosynthesis

Aluminum (0.74 mm) was found to retard bacterial growth, and enhance porphyrin formation and excretion in Arthrobacter aurescens RS-2. Coproporphyrin III was shown to be the main porphyrin excreted by aluminum-exposed A. aurescens RS-2 cultures and by RS-2 cultures grown under anoxic conditions. Synthesis and excretion of porphyrins in A. aurescens RS-2 increased in a dose-dependent manner when the bacteria were exposed to increasing aluminum concentrations. Incubation of A. aurescens RS-2 with -aminolevulinic acid (-ALA, 1.2 mm) brought about the intense formation and excretion of porphyrins by the cells, in the presence or absence of aluminum. -ALA slightly enhanced the toxicity of aluminum towards RS-2 bacteria. Furthermore, the intracellular concentration of heme was reduced by 63.9 ± 8.67% in aluminum-exposed RS-2 bacteria when compared with control cultures. The results are discussed in light of the recent finding concerning aluminum toxicity and porphyrin biosynthesis in microorganisms.     

The role of vanadium in green plants

In a series of experiments, it is demonstrated that the trace element vanadium (4·10^-7 g-at/l as NH₄VO₃) has a considerable positive influence on the synthesis of -aminolevulinic acid (-ALA) in the autotrophically growing green algaChlorella pyrenoidosa, the effect being visible by an enhanced output of the amino acid into the culture medium in presence of levulinic acid (LA). The level of intracellularly accumulated -ALA, however, is not changed in presence of the metal. The V-effect on exogenous found -ALA is suppressed, when LA is added to the nutrient medium at low pH (pH 5), although V-uptake into the algal cells is not disturbed by LA. As demonstrated in culture media with various nitrogen sources (urea, partially hydrolized urea, ammonium salts), the development of the pH during the cultivation time is important for the presentation of the V-effect on -ALA. It is suggested that vanadium acts as a catalyst in the conversion of 4,5-dioxovaleric acid to -ALA by transamination.Abbreviations -ALA -aminolevulinic acid - LA levulinic acid - DOVA 4,5-dioxovaleric acid     

Specific inhibition of tetrapyrrole biosynthesis by 3-amino 2,3-dihydrobenzoic acid (gabaculin) in the cyanobacteriumSynechococcus 6301

Gabaculin (3-amino 2,3-dihydrobenzoic acid) inhibited the growth of cyanobacteria but not of other prokaryotes. Exposure of growing cultures ofSynechococcus 6301 to 50 M gabaculin resulted in an immediate and complete inhibition of the synthesis of chlorophylla and phycocyanin. With 8 M gabaculin, tetrapyrrole synthesis was suppressed for approximately 10 h and then resumed at a lower rate than in untreated organisms. The effect of 50 M gabaculin was reversed by transferring organisms to inhibitor-free medium; chlorophylla synthesis began within 5 h and exponential growth was re-established after about 25 h. Compared with 4,6-dioxoheptanoic acid (DA) and laevulinic acid (LA), gabaculin was a much more potent inhibitor of tetrapyrrole synthesis inSynechococcus 6301. The catalytic activity of -aminolaevulinic acid (ALA) dehydratase in vitro was inhibited by DA and LA but not by 1 mM gabaculin. However, the specific activity of the dehydratase was much lower in organisms exposed to the inhibitor for 36 h. Growing cultures and cell suspensions ofSynechococcus 6301 exposed to DA excreted appreciable quantities of ALA. In contrast, relatively small amounts of ALA accumulated in the presence of gabaculin alone and this inhibitor blocked the excretion of ALA caused by DA. This suggests that the primary effect of gabaculin is the specific inhibition of the C₅ pathway for the biosynthesis of ALA.Abbreviations ALA -aminolaevulinic acid - DA 4,6-dioxoheptanoic acid - LA laevulinic acid - GABA -aminobutyric acid     

Arsenic induced blood and brain oxidative stress and its response to some thiol chelators in rats

Chronic arsenic toxicity is a widespread problem, not only in India and Bangladesh but also in various other regions of the world. Exposure to arsenic may occur from natural or industrial sources. The treatment that is in use at present employs administration of thiol chelators, such as meso 2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercaptopropane 1-sulfonate (DMPS), which facilitate its excretion from the body. However, these chelating agents are compromised with number of limitations due to their lipophobic nature, particularly for their use in cases of chronic poisoning. During chronic exposure, arsenic gains access into the cell and it becomes mandatory for a drug to cross cell membrane to chelate intracellular arsenic. To address this problem, analogs of DMSA having lipophilic character, were examined against chronic arsenic poisoning in experimental animals. In the present study, therapeutic efficacy of meso 2,3-dimercaptosuccinic acid (DMSA), sodium 2,3-dimercaptopropane 1-sulfonate (DMPS), monoisoamyl DMSA (MiADMSA) were compared in terms of reducing arsenic burden, as well as recovery in the altered biochemical variables particularly suggestive of oxidative stress. Adult male Wistar rats were given 100-ppm arsenic for 10 weeks followed by chelation therapy with the above chelating agents at a dose of 50 mg/Kg (orally) once daily for 5 consecutive days. Arsenic exposure resulted in marked elevation in reactive oxygen species (ROS) in blood, inhibition of ALAD activity and depletion of GSH. These changes were accompanied by significant decline in blood hemoglobin level. MiADMSA was the most effective chelator in reducing ROS in red blood cells, and in restoring blood ALAD compared to two other chelators. Brain superoxide dismutase (SOD) and glutathione peroxidase (GPx) decreased, while ROS and TBARS increased significantly following arsenic exposure. There was a significant increase in the activity of glutathione-S-transferase (GST) with a corresponding decline in its substrate i.e. glutathione. Among all the three chelators, MiADMSA showed maximum reduction in the level of ROS in brain. Additionally, administration of MiADMSA was most effective in counteracting arsenic induced inhibition in brain ALAD, SOD and GPx activity. Based on these results and in particular higher metal decorporation from blood and brain, we suggest MiADMSA to be a potential drug of choice for the treatment of chronic arsenic poisoning. However, further studies are required for the choice of appropriate dose, duration of treatment and possible effects on other major organs.     

Transgenic rescue for characterizing orphan receptors: a review of δ2 glutamate receptor

Even though the genomes of several major species have been sequenced, many orphan receptors with unknown ligands and mechanisms of action remain in the CNS. The 2 glutamate receptor (GluR2) is one of such receptors expressed predominantly in the cerebellar Purkinje cells. On the basis of amino acid similarity, it belongs to ionotropic glutamate receptor (iGluR) family, which mediates fast excitatory neurotransmission in the mammalian CNS. Although its null-mutant mice show prominent motor discoordination, the mechanisms by which GluR2 participates in the cerebellar functions have been unclear. To gain insight into GluR2s mechanisms, we recently generated mice that express either a wild-type or a mutant GluR2 transgene, in which the conserved arginine in GluR2s N-terminal putative ligand-binding motif was disrupted. By breeding these transgenic mice onto a GluR2^–/– background, we obtained two transgenic rescue lines. Surprisingly, the mutant GluR2 transgene was as effective as the wild-type GluR2 in rescuing the GluR2-null mice. As the disrupted arginine residue is highly conserved from ancestral bacterial periplasmic amino acid-binding proteins to mammalian iGluRs, we propose that GluR2 may not require glutamate-like amino acids and may function in an unconventional manner. This transgenic rescue approach to investigating orphan receptors is a relatively easy but powerful method when a knockout mouse with a distinct phenotype is already available. The advantages and limitations of this approach, together with certain cautions in interpreting the resulting data, are discussed in this review.     

Recorders of reef environment histories: stable isotopes in corals,giant clams,and calcareous algae

Time-series ¹⁸O and ¹³C records from cohabiting massive coralPorites australiensis and giant clamTridacna gigas from the Great Barrier Reed of Australia, and from calcareous green algae in a core through modernHalimeda bioherm accreting in the eastern Java Sea, provide insights into the complex links between environmental factors and stable isotopes imprinted in these reef skeletal materials. The aragonitic coral and giant clam offer 20 years and 15 years of growth history, respectively. The giant clam yields mean ¹⁸O and ¹³C values of-0.5±0.5 and 2.2±0.2 (n=67), which agree well with the predicted equilibrium values. The coral yields mean ¹⁸O and ¹³C values of-5.6±0.5 and-1.8±0.7 (n=84), offering a striking example of kinetic and metabolic fractionation effects. Although both the coral and giant clam harbor symbionts and were exposed to a uniform ambient environment during their growth histories, their distinct isotopic compositions demonstrate dissimilar calcification pathways. The ¹⁸O records contain periodicities corresponding to the alternating annual density bands revealed by X-radiography and optical transmitted light. Attenuation of the ¹⁸O seasonal amplitudes occurring in the giant clam record 8 years after skeletal growth commenced is attributed to a changeover from fast to slow growth rates. Extreme seasonal ¹⁸O amplitudes of up to 2.2 discerned in both the coral and giant clam records exceed the equivalent seasonal temperature contrast in the reef environment, and are caused by the combined effect of rainfall and evaporation during the monsoon and dry seasons, respectively. Thus in addition of being useful temperature recorders, reef skeletal material of sufficient longevity, such asPorites andTridacna, may also indicate rainfall variations. Changing growth rates, determined from the annual growth bands, may exert a primary control on the coral ¹³C record which shows a remarkable negative shift of 1.7 over its growth history, by comparison with only 0.15 negative shift in the contemporaneous giant clam record. Use of coral ¹³C records as proxies of fossil fuel CO₂ uptake by the ocean must be regarded with caution. The ¹⁸O and ¹³C records fromHalimeda are remarkably uniform over 1000 years of bioherm accretion history (¹⁸O=-1.7±0.2; ¹³C=3.9±0.1,n=28), in spite of variable Mg-calcite cements present in the utricles. Most of the cement infilling is probably syndepositional, and both theHalimeda aragonite and the Mg-calcite cements containign 12.3 mole % MgCO₃ are deposited in isotopic equilibrium. Therefore, in favorable circumstances these algal skeletal remains may act as the shallow water analogs of benthic foraminifera in deep sea sediments in recording ambient sea water isotopic composition and temperature.     

Inhibition of chlorophyll synthesis inHordeum vulgare by 3-amino 2,3-dihydrobenzoic acid (gabaculin)

Gabaculin (3-amino 2,3-dihydrobenzoic acid) is shown to be a very potent inhibitor of chlorophyll formation inHordeum vulgate. Exposure of leaf segments to 30/M gabaculin results in an 80% inhibition of chlorophyll synthesis, and this is paralleled by a decrease in carotenoid. Dual-inhibitor studies with dioxoheptanoic acid, which is an inhibitor of inolaevulinic acid dehydratase, show that gabaculin inhibits an earlier step than dioxoheptanoic acid and affects -aminolaevulinic acid synthesis rather than its subsequent metabolism.     

Organic nitrogen metabolism of phototrophic bacteria

Recent reviews dealing with phototrophic bacteria are concerned with bioenergetics, nitrogen fixation and hydrogen metabolism, synthesis of the photosynthetic apparatus and phylogeny/taxonomy. The organic N-metabolism of these phylogenetically diverse bacteria has last been reviewed in 1978. However, amino acid utilization and biosynthesis, ammonia assimilation, purine and pyrimidine metabolism and biosynthesis of -aminolevulinic acid as precursor of bacteriochlorophylls and hemes are topics of vital importance. This review focusses on utilization of amino acids as N- and C/N-sources, the pathways of purine and pyrimidine degradation, novel aspects of amino acid biosynthesis (with emphasis on branched-chain amino acids and -aminole-vulinic acid) and some aspects of ammonia assimilation and glutamate synthesis by purple bacteria, green sulfur bacteria and Chloroflexus aurantiacus.Abbreviations R Rhodospirillum - Rhb Rhodobacter - Rc Rhodocyclus - Rp Rhodopila - Rps Rhodopseudomonas     

Stimulation of crystallin synthesis in embryonic brain cells in culture

Summary Expression of -crystallin, a lens-specific protein, in 6-day-old chick embryonic brain cells was examined in situ and in vitro. The presence of minute amounts of -crystallin and its mRNA (-mRNA) in brain cells in situ was demonstrated by immunoblot and Northern blot analysis. In spreading cultures of the brain cells, -crystallin and -mRNA showed a significant increase from their in situ level. Immunohistological staining (peroxidase antiperoxidase) with monospecific anti-serum against -crystallin revealed that -producers were both epithelial cells and dendritic cells. Neither lentoidogenesis nor -crystallin expression was observed. Stimulation of -crystallin synthesis in cultured brain cells differed when compared with transdifferentiating cultures of neural retina cells. In the latter, -crystallin synthesis occurred concomitantly with differentiation of morphologically distinct lens cells containing -crystallin.     

Unexpected Transformations of Natural Chlorins under Treatment with Dichlorodicyanobenzoquinone

The treatment of natural chlorins with 2,3-dichloro-5,6-dicyanobenzoquinone resulted not only in the intramolecular cyclization of the propionic acid residue in position 17 with the formation of an additional -lactone cycle at the pyrrole ring D, but also in the oxygen-assisted oxidation of 8-ethyl group in ring B to an -methoxyethyl substituent.     

Trophodynamic linkage between river runoff and coastal fishery yield elucidated by stable isotope data in the Gulf of Lions (NW Mediterranean)

The link between climate-driven river runoff and sole fishery yields observed in the Gulf of Lions (NW Mediterranean) was analysed using carbon- and nitrogen stable isotopes along the flatfish food webs. Off the Rhone River, the main terrestrial (river POM) and marine (seawater POM) sources of carbon differed in ¹³C (–26.11 and –22.36, respectively). Surface sediment and suspended POM in plume water exhibited low ¹³C (–24.38 and –24.70, respectively) that differed more from the seawater POM than from river POM, demonstrating the dominance of terrestrial material in those carbon pools. Benthic invertebrates showed a wide range in ¹⁵N (mean 4.30 to 9.77) and ¹³C (mean –23.81 to –18.47), suggesting different trophic levels, diets and organic sources. Among the macroinvertebrates, the surface (mean ¹³C –23.71) and subsurface (mean ¹³C –23.81) deposit-feeding polychaetes were particularly ¹³C depleted, indicating that their carbon was mainly derived from terrestrial material. In flatfish, ¹⁵N (mean 9.42 to 10.93) and ¹³C (mean –19.95 to –17.69) varied among species, indicating differences in food source and terrestrial POM use. A significant negative correlation was observed between the percentage by weight of polychaetes in the diet and the ¹³C of flatfish white muscle. Solea solea (the main polychaete feeder) had the lowest mean ¹³C, Arnoglossus laterna and Buglossidium luteum (crustacean, mollusc and polychaete feeders) had intermediate values, and Solea impar (mollusc feeder) and Citharus linguatula (crustacean and fish feeder) exhibited the highest ¹³C. Two different benthic food webs were thus identified off the Rhone River, one based on marine planktonic carbon and the other on the terrestrial POM carried by the river. Deposit-feeding polychaetes were responsible for the main transfer of terrestrial POM to upper trophic levels, linking sole population dynamics to river runoff fluctuations.     

Photoinactivation of δ-aminolevulinic acid dehydratase from maize by flavin mononuclotide

The -aminolevulinic acid dehydratase activity was irreversibly inactivated by irradiation of the enzyme in presence of flavin mononucleotide. The loss of enzyme activity was dependent on time of irradiation, concentration of FMN and intensity of irradiance. It required oxygen and was markedly enhanced in heavy water. The presence of levulinic acid (a competitive inhibitor of -ALAD) during irradiation prevented the inactivation considerably indicating photooxidative damage at or near the active site. Superoxide dismutase, sodium benzoate and sodium formate offered no protection, but singlet oxygen quenchers like azide and tryptophan were effective. NADH, electron donor to excited flavins, also prevented the loss of enzyme activity. These results indicate that singlet oxygen produced by light absorption of FMN was responsible for the photooxidative inhibition of the enzyme.Abbreviations ALAD -aminolevulinic acid dehydratase - FMN flavin mononucleotide - O₂ ^- superoxide - H₂O₂ hydrogen peroxide - 10₂ singlet oxygen - LA levulinic acid - PBG porphobilinogen - BSA bovine serum albumin - BME 2-mercaptoethanol - SOD superoxide dismutase - pHMB para-hydroxymercuribenzoate - DTT dithiothreitol - FAD flavin adenine dinucleotide - NADH nicotinamide adenine dinucleotide     

Stable isotope ratios of Antarctic petrel (Thalassoica antarctica) and snow petrel (Pagodroma nivea) bone collagen

This study investigated the trophic hierarchy status of the Antarctic petrel (Thalassoica antarctica) and snow petrel (Pagodroma nivea) within the Southern Ocean food-web off Dronning Maud Land, Antarctica. During 1991/92 ten Antarctic petrel and ten snow petrel carcasses were collected from Jekselen (71°59S, 02°35W) and Robertskollen (71°28S, 03°15W) respectively, in the northern Ahlmannryggen, Dronning Maud Land. Collagen from the dense bone of the humeri of these carcasses was extracted and the stable carbon and nitrogen isotope ratios of these samples determined. The snow petrel bone collagen samples displayed a mean ¹³C value of –23.9±0.7 (range: –24.7 to –22.9; n=9) and a mean ¹⁵N value of 15.2±1.6 (range: 12.9 to 18.4; n=10). The corresponding values for Antarctic petrel bone collagen were –24.8±1.0 (range: –26.2 to –22.8; n=9) and 13.2±0.6 (range: 12.5 to 13.9; n=8) respectively. The difference between the species ¹³C values may indicate differences in their foraging habitat. It has previously been suggested that the snow petrel has a higher wing loading than other Procellariiforme species, making the snow petrel less adapted to pelagic foraging than related species and more likely to forage close to the sea ice edge. Algae growing under sea ice apparently can have comparatively high ¹³C values, possibly due to growing under carbon dioxide limited conditions. If so, animals foraging close to the sea ice edge might be expected to show higher ¹³C values in their body tissues than animals foraging farther out over the open ocean. However, the high ¹³C of snow petrel collagen is possibly more likely to be related to the correspondingly high ¹⁵N values found in this tissue, and hence caused by snow petrels including offal from high trophic level Antarctic mammals and birds in their diet.     

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

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