High-potential iron-sulfur protein (HiPIP) is the major electron donor to the reaction center complex in photosynthetically growing cells of the purple bacterium Rubrivivax gelatinosus

A gene encoding the high-potential iron-sulfur protein (HiPIP) was cloned from the purple photosynthetic bacterium Rubrivivax gelatinosus. An insertional disruption of this gene by a kanamycin resistance cartridge resulted in a significant decrease in the growth rate under photosynthetic growth conditions. Flash-induced kinetic measurements showed that the rate of reduction of the photooxidized reaction center is greatly diminished in the mutant depleted in the HiPIP. On the other hand, mutants depleted in the low- and high-potential cytochromes c(8), the two other soluble electron carriers, which have been shown to donate an electron to the reaction center in Rvi. gelatinosus, showed growth rates similar to those of the wild type under both photosynthetic and respiratory growth conditions. It was concluded that HiPIP is the major physiological electron donor to the reaction center in Rvi. gelatinosus cells grown under photosynthetic conditions.     

Dark aerobic growth conditions induce the synthesis of a high midpoint potential cytochrome c8 in the photosynthetic bacterium Rubrivivax gelatinosus

In several strains of the photosynthetic bacterium Rubrivivax gelatinosus, the synthesis of a high midpoint potential cytochrome is enhanced 4-6-fold in dark aerobically grown cells compared with anaerobic photosynthetic growth. This observation explains the conflicting reports in the literature concerning the cytochrome c content for this species. This cytochrome was isolated and characterized in detail from Rubrivivax gelatinosus strain IL144. The redox midpoint potential of this cytochrome is +300 mV at pH 7. Its molecular mass, 9470 kDa, and its amino acid sequence, deduced from gene sequencing, support its placement in the cytochrome c8 family. The ratio of this cytochrome to reaction center lies between 0.8 and 1 for cells of Rvi. gelatinosus grown under dark aerobic conditions. Analysis of light-induced absorption changes shows that this high-potential cytochrome c8 can act in vivo as efficient electron donor to the photooxidized high-potential heme of the Rvi. gelatinosus reaction center.     

Isolation of pigmentation mutants of the green filamentous photosynthetic bacterium Chloroflexus aurantiacus.

Mutants deficient in the production of bacteriochlorophyll c (Bchl c) and one mutant lacking colored carotenoids were isolated from the filamentous gliding bacterium Chloroflexus aurantiacus. Mutagenesis was achieved by using UV radiation or N-methyl-N'-nitro-N-nitrosoguanidine. Several clones were isolated that were deficient in Bchl c synthesis. All reverted. One double mutant deficient both in Bchl c synthesis and in the synthesis of colored carotenoids under anaerobic conditions was isolated. Isolation of a revertant in Bchl c synthesis from this double mutant produced a mutant strain of Chloroflexus that grew photosynthetically under anaerobic conditions and lacked colored carotenoids. Analysis of pigment contents and growth rates of the mutants revealed a positive association between growth rate and content of Bchl c under light-limiting conditions.     

Inhibition of porphyrin biosynthesis by exogenous 5-aminolevulinic acid in an aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

Exogenously administered 5-aminolevulinic acid (ALA) inhibited the formation of bacteriochlorophyll a (Bchl a) in a dose-dependent manner in the aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114, under dark growth conditions. The ALA concentration required for half-inhibition after 24-h growth was estimated to be about 3.0 mM. Porphyrin and Bchl precursors were not found in either the cells or the growth medium. The same inhibition was also observed with cytochrome c formation. When ALA was incubated with intact cells, a large amount of ALA was converted to an unknown metabolite. The pH optimum of the conversion was 7.8. The metabolite did not react with Ehrlich's reagent, but did so with ninhydrin, giving a yellow color. Based on analyses by several techniques including mass spectrometry, ir spectrometry, and paper electrophoresis, it was identified as 4-hydroxy-5-aminovaleric acid (HAVA). Authentic HAVA prepared from ALA was a competitive inhibitor of the enzyme, porphobilinogen synthase of Erythrobacter. The Ki value for authentic HAVA was calculated to be 2.4 mM from a Dixon plot and the HAVA concentration required for half-inhibition was 17 mM. It is concluded that in Erythrobacter cells, exogenous ALA is converted to the metabolite, HAVA, which is responsible for the inhibition of porphobilinogen synthase as well as that of Bchl a and cytochrome formation.     

Formation of the isocyclic ring of chlorophyll by isolated Chlamydomonas reinhardtii chloroplasts

Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chl biosynthesis, S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) from Mg-protoporphyrin IX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a ₅ formation by the coupled enzyme system required O₂ and was stimulated by the addition of NADP⁺, an NADPH regenerating system, and S-adenosyl-l-methionine. Product was formed at a relatively steady rate for at least 60 min.Abbreviations MgDVP Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) - SAM S-adenosyl-l-methionine     

Aerobic anoxygenic photosynthesis in Roseobacter clade bacteria from diverse marine habitats

The marine Roseobacter clade comprises several genera of marine bacteria related to the uncultured SAR83 cluster, the second most abundant marine picoplankton lineage. Cultivated representatives of this clade are physiologically heterogeneous, and only some have the capability for aerobic anoxygenic photosynthesis, a process of potentially great ecological importance in the world's oceans. In an attempt to correlate phylogeny with ecology, we investigated the diversity of Roseobacter clade strains from various marine habitats (water samples, biofilms, laminariae, diatoms, and dinoflagellate cultures) by using the 16S rRNA gene as a phylogenetic marker gene. The potential for aerobic anoxygenic photosynthesis was determined on the genetic level by PCR amplification and sequencing of the pufLM genes of the bacterial photosynthesis reaction center and on the physiological level by detection of bacteriochlorophyll (Bchl) a. A collection of ca. 1,000 marine isolates was screened for members of the marine Roseobacter clade by 16S rRNA gene-directed multiplex PCR and sequencing. The 42 Roseobacter clade isolates found tended to form habitat-specific subclusters. The pufLM genes were detected in two groups of strains from dinoflagellate cultures but in none of the other Roseobacter clade isolates. Strains within the first group (the DFL-12 cluster) also synthesized Bchl a. Strains within the second group (the DFL-35 cluster) formed a new species of Roseovarius and did not produce Bchl a under the conditions investigated here, thus demonstrating the importance of genetic methods for screening of cultivation-dependent metabolic traits. The pufL genes of the dinoflagellate isolates were phylogenetically closely related to pufL genes from Betaproteobacteria, confirming similar previous observations which have been interpreted as indications of gene transfer events.     

The Effect of Toxic Doses of Copper upon Respiration, Photosynthesis and Growth of Chlorella vulgaris

When cells of Chlorella vulgaris absorb copper under anaerobic conditions, subsequent respiration, photosynthesis and growth of the cells are all severely inhibited. This does not occur when the metal is absorbed under aerobic conditions. When, after aerobic absorption of copper, the cells are exposed to a period of anaerobiosis, respiratory inhibition is as profound as when the uptake is anaerobic. In this case, however, respiration must eventually recover, for growth is not affected so severely as it is when copper is taken up under anaerobic conditions. It is concluded that the extra copper absorbed under anaerobic conditions is directly or indirectly responsible for the greatly increased toxicity to growth, and that this copper is bound to sites not normally available under aerobic conditions. Some aspects of the apparently unique toxic effect of copper suggest that these extra sites are sulphydryl groups.     

The constant gene orf14.9, which belongs to the variable eps (exopolysaccharide) cluster, is involved in the cell growth of Streptococcus thermophilus

In Streptococcus thermophilus, the eps clusters involved in exopolysaccharide (EPS) biosynthesis are very polymorphic, nevertheless they all contain a highly conserved sequence corresponding to that of orf14.9. This open reading frame (ORF) is transcribed in a reverse direction with respect to eps genes. Amino acid sequence analysis showed a possible transmembrane location of the putative Orf14.9 protein but did not permit a proposed function. Insertional mutants of orf14.9 were obtained in strains NST2280 and A054 of S. thermophilus. EPS yields of these mutants are similar to those of their respective wild strains, suggesting that orf14.9 does not modify the quantity of produced EPS. Growth parameter determination for wild strains and their respective mutants showed that orf14.9 is involved in the cell growth of S. thermophilus.     

Control of bacteriochlorophyll accumulation by light in an aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

The effect of light on bacteriochlorophyll (Bchl) accumulation as well as the activity of two enzymes in the initial step of the tetrapyrrole biosynthetic pathway was examined in an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114. Light clearly regulated the Bchl and carotenoid accumulation, completely suppressing their levels at high light intensity. However, porphyrin and Bchl precursors were not found in either the cells or the growth medium of lighted culture. The level of Bchl showed an inverse relationship to the light energy flux. Kinetic studies showed a Hill coefficient of n = 3.3 (r = 0.973), indicating a positive cooperativity. Bchl accumulation was stopped immediately upon illumination without any lag or overshoot. Despite low Bchl content, the activities of 5-aminolevulinic acid synthetase and porphobilinogen synthase were rather stimulated, but not suppressed by light. The high activity of enzymes coincided with the results that heme contents, particularly cytochrome c and catalase activity, were increased in light-grown cells. These results suggest that light regulated Bchl accumulation, but not Bchl biosynthesis and that the effect of light is to render newly formed pigment molecules unstable.     

Inactivation of a Synechocystis sp strain PCC 6803 gene with homology to conserved chloroplast open reading frame 184 increases the photosystem II-to-photosystem I ratio.

A gene of the unicellular cyanobacterium Synechocystis sp strain PCC 6803 that is homologous to the conserved chloroplast open reading frame orf184 has been cloned and sequenced. The nucleotide sequence of the gene predicts a protein of 184 amino acids with a calculated molecular mass of 21.5 kD and two membrane-spanning regions. Amino acid sequence analysis showed 46 to 37% homology of the cyanobacterial orf184 with tobacco orf184, rice orf185, liverwort orf184, and Euglena gracilis orf206 sequences. Two orf184-specific mutants of Synechocystis sp PCC 6803 were constructed by insertion mutagenesis. Cells of mutants showed growth characteristics similar to those of the wild type. Their pigment composition was distinctly different from the wild type, as indicated by an increase in the phycocyanin-to-chlorophyll ratio. In addition, mutants also had a two- to threefold increase in photosynthetic electron transfer rates as well as in photosystem II-to-photosystem I ratio-a phenomenon hitherto not reported for mutants with altered photosynthetic characteristics. The observed alterations in the orf184-specific mutants provide strong evidence for a functional role of the orf184 gene product in photosynthetic processes.     

Physiological functions of hydroperoxidases in Rhodobacter capsulatus.

Rhodobacter capsulatus J1 has two hydroperoxidases: a catalase-peroxidase and a peroxidase. A mutant strain, AH18, that had no catalase-peroxidase was isolated. The growth rate under aerobic and photosynthetic conditions, respiration, superoxide dismutase and peroxidase activities, and pigment content of the mutant were similar to those of the wild type. AH18 was more susceptible to killing and to inhibition of nitrogenase by H2O2 but not by molecular oxygen. The incidences of spontaneous mutations were similar in both strains. Viable counts in aerobic but not anaerobic cultures of AH18 started to decline as soon as the cultures reached the stationary phase, and the rate of cell death was much higher in AH18 than in the wild type. It is inferred that the peroxidase provides protection against H2O2 in log-phase cells and that the catalase-peroxidase provides protection under the oxidative conditions that prevail in aging cultures. This protective function might be related to the dual activity of the latter as a catalase and a peroxidase or to its capacity to oxidize NADH, NADPH, and cytochrome c.     

Differential regulation of periplasmic nitrate reductase gene (napKEFDABC) expression between aerobiosis and anaerobiosis with nitrate in a denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans

A denitrifying phototroph, Rhodobacter sphaeroides f. sp. denitrificans, has the ability to denitrify by respiring nitrate. The periplasmic respiratory nitrate reductase (Nap) catalyses the first step in denitrification and is encoded by the genes, napKEFDABC. By assaying the ss-galactosidase activity of napKEFD-lacZ fusions in wild type and nap mutant cells grown under various growth conditions, the environmental signal for inducing nap expression was examined. Under anoxic conditions with nitrate, nap genes expression in the wild-type strain was highest in the dark, and somewhat lowered by incident light, but that of the napA, napB, and napC mutant strains was low, showing that nap expression is dependent on nitrate respiration. Under oxic conditions, both the wild type and nap mutant cells showed high ss-galactosidase activities, comparable to the wild-type grown under anoxic conditions with nitrate. Myxothiazol, a specific inhibitor of the cytochrome bc (1) complex, did not affect the beta-galactosidase activity in the wild-type cells grown aerobically, suggesting that the redox state of the quinone pool was not a candidate for the activation signal for aerobic nap expression. These results suggested that the trans-acting regulatory signals for nap expression differ between anoxic and oxic conditions. Deletion analysis showed that the nucleotide sequence from -135 to -88 with respect to the translational start point is essential for nap expression either under anoxic or oxic conditions, suggesting that the same cis-acting element is involved in regulating nap expression under either anoxic with nitrate or oxic conditions.     

Aerobic metabolism in the genus Lactobacillus: impact on stress response and potential applications in the food industry

This review outlines the recent advances in the knowledge on aerobic and respiratory growth of lactic acid bacteria, focusing on the features of respiration‐competent lactobacilli. The species of the genus Lactobacillus have been traditionally classified as oxygen‐tolerant anaerobes, but it has been demonstrated that several strains are able to use oxygen as a substrate in reactions mediated by flavin oxidases and, in some cases, to synthesize a minimal respiratory chain. The occurrence of genes related to aerobic and respiratory metabolism and to oxidative stress response apparently correlates with the taxonomic position of lactobacilli. Members of the ecologically versatile Lactobacillus casei, L. plantarum and L. sakei groups are apparently best equipped to deal with aerobic/respiratory growth. The shift from anaerobic growth to aerobic (oxygen) and/or respiratory promoting (oxygen, exogenous haem and menaquinone) conditions offers physiological advantages and affects the pattern of metabolite production in several species. Even if this does not result in dramatic increases in biomass production and growth rate, cells grown in these conditions have improved tolerance to heat and oxidative stresses. An overview of benefits and of the potential applications of Lactobacillus cultures grown under aerobic or respiratory conditions is also discussed.     

Selective and non-selective autophagic degradation of mitochondria in yeast

Mitochondria are essential to oxidative energy production in aerobic eukaryotic cells, where they are also required for multiple biosynthetic pathways to take place. Mitochondrial homeostasis also plays a crucial role in ageing and programmed cell death, and recent data have suggested that mitochondria degradation is a strictly regulated process. Autophagy is an evolutionary conserved mechanism that provides cells with a mechanism for the continuous turnover of damaged and obsolete macromolecules and organelles. In this work, we investigated mitochondria degradation by autophagy. Electron microscopy observations of yeast cells submitted to nitrogen starvation after growth on different carbon sources provided evidence that microautophagy, rather than macroautophagy, preferentially occurred in cells grown under nonfermentable conditions. The observation of mitochondria degradation showed that both a selective process and a nonselective process of mitochondria autophagy occurred successively. In a yeast strain inactivated for the gene UTH1, the selective process was not observed.     

Deletion or overexpression of mitochondrial NAD+ carriers in Saccharomyces cerevisiae alters cellular NAD and ATP contents and affects mitochondrial metabolism and the rate of glycolysis

The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.     

Aerobic Anoxygenic Photosynthesis in Roseobacter Clade Bacteria from Diverse Marine Habitats

The marine Roseobacter clade comprises several genera of marine bacteria related to the uncultured SAR83 cluster, the second most abundant marine picoplankton lineage. Cultivated representatives of this clade are physiologically heterogeneous, and only some have the capability for aerobic anoxygenic photosynthesis, a process of potentially great ecological importance in the world's oceans. In an attempt to correlate phylogeny with ecology, we investigated the diversity of Roseobacter clade strains from various marine habitats (water samples, biofilms, laminariae, diatoms, and dinoflagellate cultures) by using the 16S rRNA gene as a phylogenetic marker gene. The potential for aerobic anoxygenic photosynthesis was determined on the genetic level by PCR amplification and sequencing of the pufLM genes of the bacterial photosynthesis reaction center and on the physiological level by detection of bacteriochlorophyll (Bchl) a. A collection of ca. 1,000 marine isolates was screened for members of the marine Roseobacter clade by 16S rRNA gene-directed multiplex PCR and sequencing. The 42 Roseobacter clade isolates found tended to form habitat-specific subclusters. The pufLM genes were detected in two groups of strains from dinoflagellate cultures but in none of the other Roseobacter clade isolates. Strains within the first group (the DFL-12 cluster) also synthesized Bchl a. Strains within the second group (the DFL-35 cluster) formed a new species of Roseovarius and did not produce Bchl a under the conditions investigated here, thus demonstrating the importance of genetic methods for screening of cultivation-dependent metabolic traits. The pufL genes of the dinoflagellate isolates were phylogenetically closely related to pufL genes from Betaproteobacteria, confirming similar previous observations which have been interpreted as indications of gene transfer events.