Temperature and Carbon Assimilation Regulate the Chlorosome Biogenesis in Green Sulfur Bacteria

Green photosynthetic bacteria adjust the structure and functionality of the chlorosome—the light-absorbing antenna complex—in response to environmental stress factors. The chlorosome is a natural self-assembled aggregate of bacteriochlorophyll (BChl) molecules. In this study, we report the regulation of the biogenesis of the Chlorobaculum tepidum chlorosome by carbon assimilation in conjunction with temperature changes. Our studies indicate that the carbon source and thermal stress culture of C. tepidum grows slower and incorporates fewer BChl c in the chlorosome. Compared with the chlorosome from other cultural conditions we investigated, the chlorosome from the carbon source and thermal stress culture displays (a) smaller cross-sectional radius and overall size, (b) simplified BChl c homologs with smaller side chains, (c) blue-shifted Q_y absorption maxima, and (d) a sigmoid-shaped circular dichroism spectra. Using a theoretical model, we analyze how the observed spectral modifications can be associated with structural changes of BChl aggregates inside the chlorosome. Our report suggests a mechanism of metabolic regulation for chlorosome biogenesis.     

A New Glance on the Mechanism of Autotrophic CO2 Assimilation in Green Sulfur Bacteria

Microbiology - The autotrophic system of CO2 fixation in Chlorobaculum limnaeum strain C consists of two topologically independent enzyme complexes: the reverse tricarboxylic acid cycle (complex I)...     

Carbon Flow of Heliobacteria Is Related More to Clostridia than to the Green Sulfur Bacteria

The recently discovered heliobacteria are the only Gram-positive photosynthetic bacteria that have been cultured. One of the unique features of heliobacteria is that they have properties of both the photosynthetic green sulfur bacteria (containing the type I reaction center) and Clostridia (forming heat-resistant endospores). Most of the previous studies of heliobacteria, which are strict anaerobes and have the simplest known photosynthetic apparatus, have focused on energy and electron transfer processes. It has been assumed that like green sulfur bacteria, the major carbon flow in heliobacteria is through the (incomplete) reductive (reverse) tricarboxylic acid cycle, whereas the lack of CO₂-enhanced growth has not been understood. Here, we report studies to fill the knowledge gap of heliobacterial carbon metabolism. We confirm that the CO₂-anaplerotic pathway is active during phototrophic growth and that isoleucine is mainly synthesized from the citramalate pathway. Furthermore, to our surprise, our results suggest that the oxidative (forward) TCA cycle is operative and more active than the previously reported reductive (reverse) tricarboxylic acid cycle. Both isotopomer analysis and activity assays suggest that citrate is produced by a putative (Re)-citrate synthase and then enters the oxidative (forward) TCA cycle. Moreover, in contrast to (Si)-citrate synthase, (Re)-citrate synthase produces a different isomer of 2-fluorocitrate that is not expected to inhibit the activity of aconitase.     

Isotope Fractionation in Photosynthetic Bacteria during Carbon Dioxide Assimilation

The δ _PDB¹³C values have been determined for the cellular constituents and metabolic intermediates of autotrophically grown Chromatium vinosum. The isotopic composition of the HCO₃^- in the medium and the carbon isotopic composition of the bacterial cells change with the growth of the culture. The δ _PDB¹³C value of the HCO₃^- in the media changes from an initial value of −6.6‰ to +8.1‰ after 10 days of bacterial growth and the δ _PDB¹³C value of the bacterial cells change from −37.5‰ to −29.2‰ in the same period. The amount of carbon isotope fractionation during the synthesis of hexoses by the photoassimilation of CO₂ has a range of −15.5‰ at time zero to −22.0‰ after 10 days. This range of fractionation compares to the range of carbon isotope fractionation for the synthesis of sugars from CO₂ by ribulose 1,5-diphosphate carboxylase and the Calvin cycle.     

A Microsensor Study of the Interaction between Purple Sulfur and Green Sulfur Bacteria in Experimental Benthic Gradients

Abstract The interaction between the purple sulfur bacterium Thiocapsa roseopersicina and the green sulfur bacterium Prosthecochloris aestuarii was studied in a gradient chamber under a 16-hours light-8-hours dark regime. The effects of interaction were inferred by comparing the final outcome of a mixed culture experiment with those of the respective axenic cultures using the same inoculation densities and experimental conditions. Densities of bacteria were deduced from radiance microprofiles, and the chemical microenvironment was investigated with O₂, H₂S, and pH microelectrodes. P. aestuarii always formed a biofilm below the maximal oxygen penetration depth and its metabolism was strictly phototrophic. In contrast, T. roseopersicina formed a bilayer in both the mixed and the axenic culture. The top layer formed by the latter organism was exposed to oxygen, and chemotrophic sulfide oxidation took place during the dark periods, while the bottom layer grew phototrophically during the light periods only. In the mixed culture, the relative density of P. aestuarii was lower than in the axenic culture, which reflects the effects of the competition for sulfide. However, the relative density of T. roseopersicina was actually higher in the mixed culture than in the corresponding axenic culture, indicating a higher growth yield on sulfide in the mixed culture experiment. Several hypotheses are proposed to explain the effects of the interaction. Received: 15 June 1998; Accepted: 18 January 1999     

Both Forward and Reverse TCA Cycles Operate in Green Sulfur Bacteria

The anoxygenic green sulfur bacteria (GSBs) assimilate CO₂ autotrophically through the reductive (reverse) tricarboxylic acid (RTCA) cycle. Some organic carbon sources, such as acetate and pyruvate, can be assimilated during the phototrophic growth of the GSBs, in the presence of CO₂ or HCO₃⁻. It has not been established why the inorganic carbonis required for incorporating organic carbon for growth and how the organic carbons are assimilated. In this report, we probed carbon flux during autotrophic and mixotrophic growth of the GSB Chlorobaculum tepidum. Our data indicate the following: (a) the RTCA cycle is active during autotrophic and mixotrophic growth; (b) the flux from pyruvate to acetyl-CoA is very low and acetyl-CoA is synthesized through the RTCA cycle and acetate assimilation; (c) pyruvate is largely assimilated through the RTCA cycle; and (d) acetate can be assimilated via both of the RTCA as well as the oxidative (forward) TCA (OTCA) cycle. The OTCA cycle revealed herein may explain better cell growth during mixotrophic growth with acetate, as energy is generated through the OTCA cycle. Furthermore, the genes specific for the OTCA cycle are either absent or down-regulated during phototrophic growth, implying that the OTCA cycle is not complete, and CO₂ is required for the RTCA cycle to produce metabolites in the TCA cycle. Moreover, CO₂ is essential for assimilating acetate and pyruvate through the CO₂-anaplerotic pathway and pyruvate synthesis from acetyl-CoA.     

Gene Expression System in Green Sulfur Bacteria by Conjugative Plasmid Transfer

Gene transfer and expression systems in green sulfur bacteria were established by bacterial conjugation with Escherichia coli. Conjugative plasmid transfer from E. coli S17-1 to a thermophilic green sulfur bacterium, Chlorobaculum tepidum (formerly Chlorobium tepidum) WT2321, was executed with RSF1010-derivative broad-host-range plasmids, named pDSK5191 and pDSK5192, that confer erythromycin and streptomycin/spectinomycin resistance, respectively. The transconjugants harboring these plasmids were reproducibly obtained at a frequency of approximately 10^-5 by selection with erythromycin and a combination of streptomycin and spectinomycin, respectively. These plasmids were stably maintained in C. tepidum cells in the presence of these antibiotics. The plasmid transfer to another mesophilic green sulfur bacterium, C. limnaeum (formerly Chlorobium phaeobacteroides) RK-j-1, was also achieved with pDSK5192. The expression plasmid based on pDSK5191 was constructed by incorporating the upstream and downstream regions of the pscAB gene cluster on the C. tepidum genome, since these regions were considered to include a constitutive promoter and a ρ-independent terminator, respectively. Growth defections of the ∆cycA and ∆soxB mutants were completely rescued after introduction of pDSK5191-cycA and -soxB that were designed to express their complementary genes. On the other hand, pDSK5191-6xhis-pscAB, which incorporated the gene cluster of 6xhis-pscA and pscB, produced approximately four times more of the photosynthetic reaction center complex with His-tagged PscA as compared with that expressed in the genome by the conventional natural transformation method. This expression system, based on conjugative plasmid, would be applicable to general molecular biological studies of green sulfur bacteria.     

Sulfur Assimilation and the Role of Sulfur in Plant Metabolism: A Survey

Sulfur occurs in two major amino-acids, cysteine (Cys) and methionine (Met), essential for the primary and secondary metabolism of the plant. Cys, as the first carbon/nitrogen-reduced sulfur product resulting from the sulfate assimilation pathway, serves as a sulfur donor for Met, glutathione, vitamins, co-factors, and sulfur compounds that play a major role in the growth and development of plant cells. This sulfur imprinting occurs in a myriad of fundamental processes, from photosynthesis to carbon and nitrogen metabolism. Cys and Met occur in proteins, with the former playing a wide range of functions in proteins catalysis. In addition, the sulfur atom in proteins forms part of a redox buffer, as for glutathione, through specific detoxification/protection mechanisms. In this review, a survey of sulfur assimilation from sulfate to Cys, Met and glutathione is presented with highlights on open questions on their respective biosynthetic pathways and regulations that derived from recent findings. These are addressed at the biochemical and molecular levels with respect to the fate of Cys and Met throughout the plant-cell metabolism.     

Diversity of Cytochrome bc Complexes: Example of the Rieske Protein in Green Sulfur Bacteria

The Rieske 2Fe2S cluster of Chlorobium limicola forma thiosulfatophilum strain tassajara was studied by electron paramagnetic resonance spectroscopy. Two distinct orientations of its g tensor were observed in oriented samples corresponding to differing conformations of the protein. Only one of the two conformations persisted after treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. A redox midpoint potential (E_m) of +160 mV in the pH range of 6 to 7.7 and a decreasing E_m (−60 to −80 mV/pH unit) above pH 7.7 were found. The implications of the existence of differing conformational states of the Rieske protein, as well as of the shape of its E_m-versus-pH curve, in green sulfur bacteria are discussed.     

Relations between Hydrogen and Sulfur Metabolism in Purple Sulfur Bacteria

Microbiology - The review considers the role of purple sulfur bacteria in the global cycles of hydrogen and sulfur, as well as the ecology and physiology of these bacteria in relation to the...     

Taurine-Sulfur Assimilation and Taurine-Pyruvate Aminotransferase Activity in Anaerobic Bacteria

We demonstrated the ability of strictly fermentative, as well as facultatively fermentative, bacteria to assimilate sulfonate sulfur for growth. Taurine (2-aminoethanesulfonate) can be utilized by Clostridium pasteurianum C1 but does not support fermentative growth of two Klebsiella spp. and two different Clostridium spp. However, the latter are able to assimilate the sulfur of a variety of other sulfonates (e.g., cysteate, 3-sulfopyruvate, and 3-sulfolactate) anaerobically. A novel taurine-pyruvate aminotransferase activity was detected in cell extracts of C. pasteurianum C1 grown with taurine as the sole sulfur source. This activity was not detected in extracts of other bacteria examined, in C. pasteurianum C1 grown with sulfate or sulfite as the sulfur source, or in a Klebsiella isolate assimilating taurine-sulfur by aerobic respiration. More common aminotransferase activities (e.g., with aspartate or glutamate as the amino donor and pyruvate, oxalacetate, or (alpha)-ketoglutarate as the amino acceptor) were present, no matter what sulfur source was used for growth. Partial characterization of the taurine-pyruvate aminotransferase revealed an optimal temperature of 37(deg)C and a broad optimal pH range of 7.5 to 9.5.     

Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum

The assimilation of NH₄⁺ causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoenolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH₄⁺ assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.     

Dual Localized AtHscB Involved in Iron Sulfur Protein Biogenesis in Arabidopsis

Background

Iron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.

Methodology/Principal Findings

In this study we demonstrate that AtHscB from Arabidopsis can rescue the Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.

Conclusions

Our data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol.     

Ammonia Assimilation in Rumen Bacteria: A Review

In the rumen bacteria, ammonia as the end product of nitrogen is incorporated into carbon skeleton (α-ketoglutarate) to yield glutamine and glutamate which are important nitrogen donors in nitrogenous compounds metabolism in cells. The enzymes glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase are involved in these processes. Some experimental results have proven that the global nitrogen regulation system may participate in the regulation of assimilation of ammonia in rumen bacteria. This review offers a current perspective on the pathways and key enzymes of ammonia assimilation in rumen bacteria with the possible molecular regulation strategy, while points out the further research direction.     

Carbon and Sulfur Metabolism in Representatives of Two Clusters of Bacteria of the Genus Leucothrix: A Comparative Study

Major pathways of carbon and sulfur metabolisms were studied in representatives of two clusters of bacteria: Leucothrix thiophila (cluster I, strains 2WS, 4WS, and 6WS) and Leucothrix sp. (cluster II, strains 1WS, 3WS, and 5WS). All strains were capable of chemoorganoheterotrophic growth, as well as of chemolithoheterotrophic growth in the presence of reduced sulfur compounds. The bacteria were found to possess a complete set of the enzymes of the tricarboxylic acid cycle and glyoxylate cycle. The dehydrogenase activity in cells of cluster I strains was an order of magnitude lower than in cluster II strains and in other known heterotrophic bacteria. Cells of bacteria of both clusters exhibited high activity levels of enzymes involved in the energy metabolism of sulfur. The oxidation of sulfur compounds and the operation of the electron-transport chain were shown to be related. Cluster II bacteria more efficiently use organic compounds in their energy metabolism, whereas cluster I bacteria are characterized by more efficient utilization of reduced sulfur compounds. During sulfite oxidation, cluster I bacteria can synthesize ATP both via substrate-level phosphorylation and oxidative phosphorylation, whereas cluster II bacteria synthesize ATP only via the latter process.     

Diversity and Detection of Nitrate Assimilation Genes in Marine Bacteria

A PCR approach was used to construct a database of nasA genes (called narB genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A nasA-specific PCR primer set that could be used to selectively amplify the nasA gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced nasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.     

Building Better Barrels – β-barrel Biogenesis and Insertion in Bacteria and Mitochondria

β-barrel proteins are folded and inserted into outer membranes by multi-subunit protein complexes that are conserved across different types of outer membranes. In Gram-negative bacteria this complex is the barrel-assembly machinery (BAM), in mitochondria it is the sorting and assembly machinery (SAM) complex, and in chloroplasts it is the outer envelope protein Oep80. Mitochondrial β-barrel precursor proteins are translocated from the cytoplasm to the intermembrane space by the translocase of the outer membrane (TOM) complex, and stabilized by molecular chaperones before interaction with the assembly machinery. Outer membrane bacterial BamA interacts with four periplasmic accessory proteins, whereas mitochondrial Sam50 interacts with two cytoplasmic accessory proteins. Despite these major architectural differences between BAM and SAM complexes, their core proteins, BamA and Sam50, seem to function the same way. Based on the new SAM complex structures, we propose that the mitochondrial β-barrel folding mechanism follows the budding model with barrel-switching aiding in the release of new barrels. We also built a new molecular model for Tom22 interacting with Sam37 to identify regions that could mediate TOM-SAM supercomplex formation.     

Ubiquitous Dissolved Inorganic Carbon Assimilation by Marine Bacteria in the Pacific Northwest Coastal Ocean as Determined by Stable Isotope Probing

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM ¹³C-NaHCO₃, doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.     

Specific Gene bciD for C7-Methyl Oxidation in Bacteriochlorophyll e Biosynthesis of Brown-Colored Green Sulfur Bacteria

The gene named bciD, which encodes the enzyme involved in C7-formylation in bacteriochlorophyll e biosynthesis, was found and investigated by insertional inactivation in the brown-colored green sulfur bacterium Chlorobaculum limnaeum (previously called Chlorobium phaeobacteroides). The bciD mutant cells were green in color, and accumulated bacteriochlorophyll c homologs bearing the 7-methyl group, compared to C7-formylated BChl e homologs in the wild type. BChl-c homolog compositions in the mutant were further different from those in Chlorobaculum tepidum which originally produced BChl c: (3¹ S)-8-isobutyl-12-ethyl-BChl c was unusually predominant.