Photoheterotrophic Conditions Research Articles

Using fluorine-induced chemiluminescence to detect organo-metalloids the headspace of phototrophic bacterial cultures amended with selenium and tellurium

Purple nonsulfur bacteria are resistant to metalloid oxyanions. The key to this resistance seems to lie in their ability to reduce and, in some cases, methylate these toxic chemical species. Six strains of purple nonsulfur bacteria have been grown under photo-heterotrophic conditions and exposed to varied concentrations of inorganic forms of selenium, tellurium and dimethyl selenone, a proposed biological intermediate of the selenium reduction/methylation process. Selenium and tellurium metallic powders have also been added to live cultures. All of the phototrophic bacteria studied here reduce and methylate one or the other oxyanion of Se used, and three strains reduce and methylate Te° and Se°. All strains respond to the addition of dimethyl selenone by producing dimethyl selenide and/or dimethyl diselenide. In all six cases the highest amounts of volatile selenium compounds are found in cultures amended with dimethyl selenone and the lowest amounts in cultures doped with selenate. Some of the phototrophic bacteria studied here, when amended with oxyanions of both Se and Te, increase their release of dimethyl telluride (produced by their bioreduction and methylation of tellurate) in the presence of selenate. Control experiments show that this synergism is biological and not merely caused by the presence of other organo-sulfides or -selenides in culture.

Production of antioxidant vitamins, β-carotene, vitamin C, and vitamin E, by two-step culture ofEuglena gracilis Z

Euglena gracilis Z is one of the few microorganisms which simultaneously produces antioxidant vitamins such as beta-carotene and vitamins C and E. Photoheterotrophically cultured E. gracilis Z produced larger levels of biomass but with a lower content of antioxidant vitamins than photoautotrophically grown cultures. For efficient production of these vitamins, a two-step culture was performed. Cells were grown photoheterotrophically and then transferred to photoautotrophic conditions. When E. gracilis Z cells were grown in fed-batch culture under photoheterotrophic conditions, their density reached 19 g/L after 145 h. Subsequent transfer of these cells to photoautotrophic conditions increased vitamin content, enhancing the total vitamin yields, which were 71.0 mg/L of beta-carotene, 30.1 mg/L of vitamin E, and 86.5 mg/L of vitamin C. (c) 1997 John Wiley & Sons, Inc.

Production of antioxidant vitamins, β‐carotene, vitamin C, and vitamin E, by two‐step culture of Euglena gracilis Z

Euglena gracilis Z is one of the few microorganisms which simultaneously produces antioxidant vitamins such as β-carotene and vitamins C and E. Photoheterotrophically cultured E. gracilis Z produced larger levels of biomass but with a lower content of antioxidant vitamins than photoautotrophically grown cultures. For efficient production of these vitamins, a two-step culture was performed. Cells were grown photoheterotrophically and then transferred to photoautotrophic conditions. When E. gracilis Z cells were grown in fed-batch culture under photoheterotrophic conditions, their density reached 19 g/L after 145 h. Subsequent transfer of these cells to photoautotrophic conditions increased vitamin content, enhancing the total vitamin yields, which were 71.0 mg/L of β-carotene, 30.1 mg/L of vitamin E, and 86.5 mg/L of vitamin C. © 1997 John Wiley & Sons, Inc.

CORRELATED BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES IN NITROGEN‐STARVED EUGLENA GRACILIS1

ABSTRACTGrowth of Euglena gracilis Z Pringsheim under photoheterotrophic conditions in a nitrogen‐deprived medium resulted in progressive loss of chloroplastic material until total bleaching of the cells occurred. Biochemical analysis and ultrastructural observation of the first stages of the starvation process demonstrated an early lag phase (from 0 to 9 h) in which cells increased in size, followed by a period of cell division, apparently supported by the mobilization of some chloroplastic proteins such as the photosynthetic CO2‐fixing enzyme ribulose‐1,5‐bisphosphate carboxylase/oxygenase. The degradation of the enzyme started after 9 h of starvation and was preceded by a transient concentration of this protein in pyrenoidal structures. Protein nitrogen and photosynthetic pigments as well as number of chloroplasts per cell decreased during proliferation through mere distribution among daughter cells. However, after 24 h, when cell division had almost ceased, there was a slow but steady decline of photosynthetic pigments. This was paralleled by observable ultrastructural changes including progressive loss of chloroplast structure and accumulation of paramylon granules and lipid globules in the cytoplasm. These findings reinforce the role of chloroplastic materials as a nitrogen source during starvation of E. gracilis in a carbon‐rich medium. The excess of ribulose‐1,5‐bisphosphate carboxylase/oxygenase acts as a first reservoir that, once exhausted, is superseded by the generalized disassembly of the photosynthetic structures, if the adverse environment persists more than 24 h.

Cloning and sequencing of the genes encoding the light-harvesting B806-866 polypeptides and initial studies on the transcriptional organization of puf2B, puf2A and puf2C in Chloroflexus aurantiacus.

The genes encoding the alpha- and beta-polypeptide subunits of the B806-866 membrane-bound light-harvesting complex of Chloroflexus aurantiacus have been cloned and the nucleotide sequences determined. The gene puf2A, which encodes the B806-866 alpha-polypeptide, began 28 bases downstream of the stop codon of puf2B, which encodes the B806-866 beta gene. The gene-encoding cytochrome c-554, puf2C, was found about 250 bp downstream of puf2A. puf2A encoded a 13 amino acid extension at the C-terminus of the B806-866 alpha-polypeptide that was not present in the mature protein. These genes, unlike those of purple nonsulfur bacteria, did not form a contiguous operon with puf1L or puf1M, the genes encoding the L and M subunits of the photochemical reaction center. The occurrence of the two latter genes and of puf2B and puf2A in two separate operons has not been observed in purple bacteria. Under photoheterotrophic growth conditions, puf2B and puf2A were encoded on an abundant mRNA that was 0.5 kb long. Two monocistronic transcripts for puf2C were observed that had different 5'-ends. One transcript encoding all three genes was also detected. Nucleotide sequences very similar to the consensus promoter sequence of the Escherichia coli RNA polymerase sigma 70 subunit were found seven and eight bases upstream of the 5'-end of mRNA encoding puf2B and for one of the monocistronic mRNA encoding puf2C, respectively.

Photoheterotrophic and chemoheterotrophic dinitrogen fixation and nitrate utilization by the cyanobacteriumAnabaena torulosa

Anabaena torulosa exhibited fructose-dependent growth, heterocyst differentiation and N2 fixation in nitrate-free (diazotrophic) cultures in photoheterotrophic and chemoheterotrophic conditions. The incorporation of nitrate into such cultures inhibited the formation of heterocysts and N2 fixation. The rate of NO 3 − uptake byA. torulosa in photoautotrophic, photoheterotrophic and chemoheterotrophic conditions was similar but it increased by 100% in phototrophic conditions. The activity of glucose-6-phosphate dehydrogenase was found to be maximum in phototrophic and photoheterotrophic conditions. Ferredoxin-NADP+ reductase, nitrate reductase and glutamate-ammonia ligase activities suggest that nitrate utilization takes place in nonphotosynthetic conditions.

Deletion of cytochrome c oxidase genes from the cyanobacterium Synechocystis sp. PCC6803: Evidence for alternative respiratory pathways

An oligonucleotide directed against a highly conserved region of aa3-type cytochrome c oxidases was used to clone the cox genes from the cyanobacterium Synechocystis sp. PCC6803. Several overlapping clones were obtained that contained the coxB, coxA, and coxC genes, transcribed in the same direction in that order, coding for subunits II, I, and III, respectively. The deduced protein sequences of the three subunits showed high sequence similarity with the corresponding subunits of all known aa3-type cytochrome c oxidases. A 1.94-kb HindII fragment containing most of coxA and about half of coxC was deleted and replaced by a cassette coding for kanamycin resistance. Mutant cells that were homozygous for the deleted cox locus were obtained. They were viable under photoautotrophic and photoheterotrophic conditions, but contained no cytochrome c oxidase activity. Nevertheless, these mutant cells showed almost normal respiration, defined as cyanide-inhibitable O2 uptake by whole cells in the dark. It is concluded, therefore, that aa3-type cytochrome c oxidase is not the only terminal respiratory oxidase in Synechocystis sp. PCC6803.

Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function.

To design an in vivo system allowing detailed analysis of photosystem II (PSII) complexes without significant interference from other pigment complexes, part of the psaAB operon coding for the core proteins of photosystem I (PSI) and part of the apcE gene coding for the anchor protein linking the phycobilisome to the thylakoid membrane were deleted from the genome of the cyanobacterium Synechocystis sp strain PCC 6803. Upon transformation and segregation at low light intensity (5 microE m-2 sec-1), a PSI deletion strain was obtained that is light tolerant and grows reasonably well under photoheterotrophic conditions at 5 microE m-2 sec-1 (doubling time approximately 28 hr). Subsequent inactivation of apcE by an erythromycin resistance marker led to reduction of the phycobilin-to-chlorophyll ratio and to a further decrease in light sensitivity. The resulting PSI-less/apcE- strain grew photoheterotrophically at normal light intensity (50 microE m-2 sec-1) with a doubling time of 18 hr. Deletion of apcE in the wild type resulted in slow photoautotrophic growth. The remaining phycobilins in apcE- strains were inactive in transferring light energy to PSII. Cells of both the PSI-less and PSI-less/apcE- strains had an approximately sixfold enrichment of PSII on a chlorophyll basis and were as active in oxygen evolution (on a per PSII basis) as the wild type at saturating light intensity. Both PSI-less strains described here are highly appropriate both for detailed PSII studies and as background strains to analyze site- and region-directed PSII mutants in vivo.

Nif gene expression studies in Rhodobacter capsulatus: nfrC‐independent repression by high ammonium concentrations

The expression of nif genes in Rhodobacter capsulatus depends on the two regulatory genes, rpoN and nifA, encoding a nif-specific alternative sigma factor of RNA polymerase and a nif-specific transcriptional activator, respectively. The expression of the rpoN gene itself is also RPON/NIFA dependent. In order to better characterize the regulation of nif gene induction, chromosomal nifH-, rpoN-, nifA1- and nifA2- lacZ fusions were constructed and the expression of these different nif-lacZ fusions was determined under photoheterotrophic conditions at different starting ammonium concentrations. The two nifA genes were found to be induced first, followed by nifH and finally by rpoN upon weak, medium and strong nitrogen starvation, respectively. This induction profile and the correlation between the expression of the different nif genes suggested that nifA1 expression is the limiting factor for nif gene induction. This hypothesis was tested by construction of different nifA1 overexpressing mutants. Contrary to the current model of nif gene expression in R. capsulatus, which predicted constitutive nif gene expression in such mutants, a strong repression of nifH and rpoN was found at high ammonium concentration. The low nifH expression under these conditions is unaffected by nifA2 and is not increased in a ntrC mutant, ruling out any role of NTRC as a mediator of this repression. This finding implies an additional, so far unidentified, regulation by fixed nitrogen in R. capsulatus. Changing the expression level of rpoN indicated that low levels of RPON are already sufficient for full nifH induction. The nifA1 and rpoN expression mutants were also tested for diazotrophic growth. Similar generation times were determined for the mutants and for the wild type, but diazotrophic growth of the nifA1 over-expressing ntrC mutant RCM14 did not start until after a prolonged lag phase of two to three days.

Effect of Glucose Utilization on Nitrite Excretion by the Unicellular Cyanobacterium Synechocystis sp. Strain PCC 6803

Up to 1 mM nitrite was excreted by Synechocystis strain 6803 cells growing under mixotrophic or photoheterotrophic conditions. This excretion is not due to a lower ratio of nitrite and nitrate reductase activities in the presence of glucose but seems to be related to a shortage of reduced ferredoxin, their electron donor, as a result of a decrease in noncyclic photosynthetic flow observed under these circumstances. Because about 60% of the reduced nitrate is excreted, the potential utilization of cyanobacteria for removal of nitrate from contaminated waters containing high concentrations of organic compounds is questioned.

Akinete differentiation in phototrophic, photoheterotrophic and chemoheterotrophic conditions inAnabaena torulosa

Akinete differentiation inAnabaena torulosa occurred under phototrophic, photoheterotrophic and chemoheterotrophic conditions at the expense of fructose in nitrate-free and nitrate media. Maximum frequency of akinetes was recorded in nitrate-free cultures in photoheterotrophic condition followed by chemoheterotrophic condition. The activity of glucose-6-phosphate dehydrogenase (GPDase) and the levels of glycogen exhibited a correlation with the presence and absence of nitrate and maximum activity was detected in photoheterotrophic condition at the maturation phase. The cells in nitrate-free medium exhibited enhanced levels of GPDase, nitrogen fixation and low levels of glycogen in the presence of fructose. A lowering of GPDase activity in nitrate grown cells was found to be associated with the accumulation of glycogen. The results presented here further suggest that a critical balance of the C:N ratio triggers akinete differentiation.

Isolation and characterization of the ndhF gene of Synechococcus sp. strain PCC 7002 and initial characterization of an interposon mutant

The ndhF gene of the unicellular marine cyanobacterium Synechococcus sp. strain PCC 7002 was cloned and characterized. NdhF is a subunit of the type 1, multisubunit NADH:plastoquinone oxidoreductase (NADH dehydrogenase). The nucleotide sequence of the gene predicts an extremely hydrophobic protein of 664 amino acids with a calculated mass of 72.9 kDa. The ndhF gene was shown to be single copy and transcribed into a monocistronic mRNA of 2,300 nucleotides. An ndhF null mutation was successfully constructed by interposon mutagenesis, demonstrating that NdhF is not required for cell viability under photoautotrophic growth conditions. The mutant strain exhibited a negligible rate of oxygen uptake in the dark, but its photosynthetic properties (oxygen evolution, chlorophyll/P700 ratio, and chlorophyll/P680 ratio) were generally similar to those of the wild type. Although the ndhF mutant strain grew as rapidly as the wild-type strain at high light intensity, the mutant grew more slowly than the wild type at lower light intensities and did not grow at all under photoheterotrophic conditions. The roles of the NADH:plastoquinone oxidoreductase in photosynthetic and respiratory electron transport are discussed.

Effect of nutritional regime on accumulation of cobalt, manganese and zinc by green microalgae

Accumulation of cobalt, manganese and zinc by the algae Chlorella emersonii, Chlamydomonas reinhardtii and Scenedesmus obliquus has been characterized under photoautotrophic, photoheterotrophic and chemoheterotrophic nutritional regimes. All three species accumulated smaller amounts of Co2+, Mn2+ and Zn2+ under chemoheterotrophic and photoheterotrophic conditions than under photoautotrophic conditions except in the case of cobalt accumulation by C. reinhardtii where there was little difference in the amount of cobalt accumulated under any of the nutritional regimes. Decreased accumulation of the three metals by C. emersonii and C. reinhardtii largely resulted from a decrease in the initial biosorptive phase of uptake whereas the decrease in Mn2+ and Zn2+ accumulation by C. reinhardtii under chemoheterotrophic and photoheterotrophic conditions was due to a decrease in the slow energy-dependent phase of uptake.

Effect of nutritional regime on accumulation of cobalt, manganese and zinc by green microalgae

Accumulation of cobalt, manganese and zinc by the algae Chlorella emersonii, Chlamydomonas reinhardtii and Scenedesmus obliquus has been characterized under photoautotrophic, photoheterotrophic and chemoheterotrophic nutritional regimes. All three species accumulated smaller amounts of Co2+, Mn2+ and Zn2+ under chemoheterotrophic and photoheterotrophic conditions than under photoautotrophic conditions except in the case of cobalt accumulation by C. reinhardtii where there was little difference in the amount of cobalt accumulated under any of the nutritional regimes. Decreased accumulation of the three metals by C. emersonii and C. reinhardtii largely resulted from a decrease in the initial biosorptive phase of uptake whereas the decrease in Mn2+ and Zn2+ accumulation by C. reinhardtii under chemoheterotrophic and photoheterotrophic conditions was due to a decrease in the slow energy-dependent phase of uptake.

Identification and Characterization of the ictA/ndhL Gene Product Essential to Inorganic Carbon Transport of Synechocystis PCC6803

The ictA gene, renamed ndhL in this paper, essential to inorganic carbon transport of Synechocystis PCC6803, was expressed in Eschericia coli as a fusion protein with glutathione S-transferase. An antibody was raised against this fusion protein. Western analysis of the thylakoid membrane of wild-type (WT) Synechocystis revealed that a protein with an apparent molecular mass of 6.7 kilodaltons cross-reacted with this antibody. No immunoreactive protein was present in the thylakoid membranes of the Synechocystis mutants, RKb and M9, which have defects in the ictA/ndhL gene, or in the cytoplasmic membranes of the WT and mutant cells. Thus, the protein reacted with the antibody is the ictA gene product (IctA) and is localized in the thylakoid membrane of WT cells. IctA was absent in the thylakoid membranes of the M55 mutant, in which the ndhB gene is inactivated, and was poorly immunostained in the membranes of the mutants (M-ndhC and M-ndhK) constructed by inactivating the ndhC and ndhK genes of WT Synechocystis, respectively. The carbon dioxide uptake activity was nearly zero in M-ndhK and was about 40% of the activity of WT cells in M-ndhC. The RKb, M-ndhC, and M-ndhK mutants were unable to grow or grew very slowly under photoheterotrophic conditions. These results indicated that NADH dehydrogenase is essential to inorganic carbon transport and photoheterotrophic growth of Synechocystis and that IctA is one of the subunits of this enzyme.

Reinvestigation of regulation of biosynthesis and subunit composition of nickel-dependent Hup-hydrogenase of Rhodospirillum rubrum

Regulation of biosynthesis and structural properties of the membrane-bound ‘hydrogen uptake’ (Hup)-hydrogenase of Rhodospirillum rubrum were studied. Under photoheterotrophic conditions, the highest activities were recorded in media with L-lactate, the lowest activities with malate. At high concentrations (20–40 mM), NH 4 + or glutamate suppressed biosynthesis of hydrogenase. In culture media supplemented with 0.5 mM EDTA, normal Hup activities were only obtained in the presence of high concentrations of Ni 2+ (80–150 μM). Activation of hydrogenase biosynthesis by Ni 2+ was completely inhibited by chloramphenicol. The enzyme, solubilized from intracytoplasmic membranes by Triton X-114 and partially purified by gelfiltration, had a M r of about 100 000. When analysed by SDS-PAGE, the native enzyme yielded three different protein bands with M r values of 40 000, 34 000 and 29 000.

Reinvestigation of regulation of biosynthesis and subunit composition of nickel-dependent Hup-hydrogenase ofRhodospirillum rubrum

Regulation of biosynthesis and structural properties of the membrane-bound ‘hydrogen uptake’ (Hup)-hydrogenase of Rhodospirillum rubrum were studied. Under photoheterotrophic conditions, the highest activities were recorded in media with L-lactate, the lowest activities with malate. At high concentrations (20–40 mM), NH4+ or glutamate suppressed biosynthesis of hydrogenase. In culture media supplemented with 0.5 mM EDTA, normal Hup activities were only obtained in the presence of high concentrations of Ni2+ (80–150 μM). Activation of hydrogenase biosynthesis by Ni2+ was completely inhibited by chloramphenicol. The enzyme, solubilized from intracytoplasmic membranes by Triton X-114 and partially purified by gelfiltration, had a Mr of about 100 000. When analysed by SDS-PAGE, the native enzyme yielded three different protein bands with Mr values of 40 000, 34 000 and 29 000.

Toxicity of phenol and three nitrophenols towards growth and metabolic activities ofNostoc linckia, isolated from soil

The toxicity of phenol and its nitrosubstituentso-nitrophenol (ONP),m-nitrophenol (MNP), andp-nitrophenol (PNP) was determined towards growth and metabolic activities of a diazotrophic cyanobacterium,Nostoc linckia, isolated from soil. Nitrophenols, but not phenol significantly inhibited the production of cell constituents (chlorophylla, protein and carbohydrate),14CO2 uptake and activities of nitrate reductase, nitrogenase, and glutamine synthetase. Growth of the organism under photoheterotrophic conditions (0.1% acetate) did not reduce the toxicity of nitrophenols. However, the addition of 10 μM adenosine triphosphate (ATP) to MNP treatments reversed the toxicity. Transmission electron microscopy revealed the secretion of mucous around the filament and induction of spore formation in the cultures subjected to nitrophenol toxicity.

Morphometric and Stereologic Analysis of Chlorella vulgaris Under Heterotrophic Growth Conditions

A computer-supported determination of stereological paramelers was used to study the possible ultrastructural changes of Chlorella vulgairis UAM 101 under photolithotrophic, mixotrophic and photoheterotrophic conditions of growth. Data recording was carried out through a semi-automatic digitizing image analysis system instead of the current method of superimposition of an array of short lines. Glucose promoted drastic physiological changes [Martinez and Orus. 1991. Plant Physiology (in press)], which strongly affected the size of the cells and volume densities of storage materials. However, volumetric ratios of the mitochondrion or chloroplast active fraction were not affected by the presence of glucose, probably indicating that these ratios are characteristic of each species. Cell wall ultrastructure was also analysed and the presence of sporopollenin demonstrated, in contrast to the thin and sporopollenin-lacking cell wall generally described for Chlorella vulgaris species.

Nitrogen fixation by Oscillatoria spp. under autotrophic and photoheterotrophic conditions

Summary: Oscillatoria spp. UCSB8 and UCSB25 are both capable of aerobic N2 fixation. The optimum temperature for C2H2 reduction was 22 °C for Oscillatoria sp. UCSB8 and 35 °C for Oscillatoria sp. UCSB25, whilst the optimum temperature for growth on N2 was 25 °C and 30 °C, respectively. In Oscillatoria sp. UCSB25, but not in UCSB8, inhibition of N2 fixation may limit diazotrophic growth at temperatures above 35 °C. When grown under alternating 12 h light and 12 h darkness, both isolates reduced C2H2 predominantly in the dark and both were capable of N2 fixation and photoheterotrophic growth in the presence of 20 μm-DCMU to inhibit photosystem II activity. Under these conditions, the best exogenous carbon source for Oscillatoria sp. UCSB8 was glucose, whilst that for Oscillatoria sp. UCSB25 was fructose. In Oscillatoria sp. UCSB8, exogenous glucose was catabolized mainly through the oxidative pentose phosphate pathway. Although cultures grown photoheterotrophically showed higher specific activities of nitrogenase than photoautotrophic cultures, they grew more slowly. Furthermore, cultures grown photoheterotrophically under alternating light and darkness reduced C2H2 both in the light and in the dark, but the highest rates of C2H2 reduction were observed in the dark. This cyclic pattern of N2 fixation was independent of photosystem II activity.