Respiration Strategies Utilized by the Gill Endosymbiont from the Host Lucinid Codakia orbicularis (Bivalvia: Lucinidae)

The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.     

Plasticity of symbiont acquisition throughout the life cycle of the shallow-water tropical lucinid Codakia orbiculata (Mollusca: Bivalvia)

In marine invertebrates that acquire their symbionts from the environment, these are generally only taken up during early developmental stages. In the symbiosis between lucinid clams and their intracellular sulfur-oxidizing bacteria, it has been shown that the juveniles acquire their symbionts from an environmental stock of free-living symbiont forms, but it is not known if adult clams are still competent to take up symbiotic bacteria from the environment. In this study, we investigated symbiont acquisition in adult specimens of the lucinid clam Codakia orbiculata, using transmission electron microscopy, fluorescence in situ hybridization, immunohistochemistry and PCR. We show here that adults that had no detectable symbionts after starvation in aquaria for 6 months, rapidly reacquired symbionts within days after being returned to their natural environments in the field. Control specimens that were starved and then exposed to seawater aquaria with sulfide did not reacquire symbionts. This indicates that the reacquisition of symbionts in the starved clams returned to the field was not caused by high division rates of a small pool of remaining symbionts that we were not able to detect with the methods used here. Immunohistochemistry with an antibody against actin, a protein involved in the phagocytosis of intracellular bacteria, showed that actin was expressed at the apical ends of the gill cells that took up symbionts, providing further evidence that the symbionts were acquired from the environment. Interestingly, actin expression was also observed in symbiont-containing cells of untreated lucinids freshly collected from the environment, indicating that symbiont acquisition from the environment occurs continuously in these clams throughout their lifetime.     

CO2 uptake and fixation by endosymbiotic chemoautotrophs from the bivalve Solemya velum

Chemoautotrophic symbioses, in which endosymbiotic bacteria are the major source of organic carbon for the host, are found in marine habitats where sulfide and oxygen coexist. The purpose of this study was to determine the influence of pH, alternate sulfur sources, and electron acceptors on carbon fixation and to investigate which form(s) of inorganic carbon is taken up and fixed by the gamma-proteobacterial endosymbionts of the protobranch bivalve Solemya velum. Symbiont-enriched suspensions were generated by homogenization of S. velum gills, followed by velocity centrifugation to pellet the symbiont cells. Carbon fixation was measured by incubating the cells with (14)C-labeled dissolved inorganic carbon. When oxygen was present, both sulfide and thiosulfate stimulated carbon fixation; however, elevated levels of either sulfide (>0.5 mM) or oxygen (1 mM) were inhibitory. In the absence of oxygen, nitrate did not enhance carbon fixation rates when sulfide was present. Symbionts fixed carbon most rapidly between pH 7.5 and 8.5. Under optimal pH, sulfide, and oxygen conditions, symbiont carbon fixation rates correlated with the concentrations of extracellular CO(2) and not with HCO(3)(-) concentrations. The half-saturation constant for carbon fixation with respect to extracellular dissolved CO(2) was 28 +/- 3 microM, and the average maximal velocity was 50.8 +/- 7.1 micromol min(-1) g of protein(-1). The reliance of S. velum symbionts on extracellular CO(2) is consistent with their intracellular lifestyle, since HCO(3)(-) utilization would require protein-mediated transport across the bacteriocyte membrane, perisymbiont vacuole membrane, and symbiont outer and inner membranes. The use of CO(2) may be a general trait shared with many symbioses with an intracellular chemoautotrophic partner.     

Nitrate Respiration in Chemoautotrophic Symbionts of the Bivalve Lucinoma aequizonata Is Not Regulated by Oxygen

The marine bivalve Lucinoma aequizonata has intracellular chemoautotrophic symbionts residing in the gill tissue. These bacteria are capable of nitrate respiration even under fully saturated oxygen conditions. Nitrate reductase in the symbionts of L. aequizonata appears to be constitutively expressed and without significant regulation by oxygen or nitrate. We discuss the stationary-phase growth state of the symbionts as an explanation for the lack of enzyme induction.     

Bacterial host specificity of Lucinacea endosymbionts: Interspecific variation in 16S rRNA sequences

Abstract Three tropical lucinid clams ( Codakia orbiculata, Codakia pectinella and Lucina nassula ) from a shallow coastal environment have been studied regarding to their thioautotrophic bacterial endosymbionts. The 16S rRNA genes (rDNA) from these three endosymbionts were amplified using PCR. Phylogenetic analysis by distance matrix and parsimony methods always placed the newly examined symbionts within the monophyletic group composed of symbionts of the bivalve superfamily Lucinacea. A same single 16S rRNA sequence was found in C. orbiculata and C. pectinella and was identical to that found in C. orbicularis and Linga pensylvanica , two other lucinids living in the same type of environment. These data indicate that a same symbiont species may be associated with different host species. Lucina nassula hosts a symbiont with a distinct 16S rDNA sequence, but very closely related to the former.     

CO2 Uptake and Fixation by Endosymbiotic Chemoautotrophs from the Bivalve Solemya velum

Chemoautotrophic symbioses, in which endosymbiotic bacteria are the major source of organic carbon for the host, are found in marine habitats where sulfide and oxygen coexist. The purpose of this study was to determine the influence of pH, alternate sulfur sources, and electron acceptors on carbon fixation and to investigate which form(s) of inorganic carbon is taken up and fixed by the gamma-proteobacterial endosymbionts of the protobranch bivalve Solemya velum. Symbiont-enriched suspensions were generated by homogenization of S. velum gills, followed by velocity centrifugation to pellet the symbiont cells. Carbon fixation was measured by incubating the cells with ¹⁴C-labeled dissolved inorganic carbon. When oxygen was present, both sulfide and thiosulfate stimulated carbon fixation; however, elevated levels of either sulfide (>0.5 mM) or oxygen (1 mM) were inhibitory. In the absence of oxygen, nitrate did not enhance carbon fixation rates when sulfide was present. Symbionts fixed carbon most rapidly between pH 7.5 and 8.5. Under optimal pH, sulfide, and oxygen conditions, symbiont carbon fixation rates correlated with the concentrations of extracellular CO₂ and not with HCO₃⁻ concentrations. The half-saturation constant for carbon fixation with respect to extracellular dissolved CO₂ was 28 ± 3 μM, and the average maximal velocity was 50.8 ± 7.1 μmol min⁻¹ g of protein⁻¹. The reliance of S. velum symbionts on extracellular CO₂ is consistent with their intracellular lifestyle, since HCO₃⁻ utilization would require protein-mediated transport across the bacteriocyte membrane, perisymbiont vacuole membrane, and symbiont outer and inner membranes. The use of CO₂ may be a general trait shared with many symbioses with an intracellular chemoautotrophic partner.     

Nitrate and Nitrite Reduction by Wolffia arrhiza

Nitrate reductase was not found to be present in or associated with partially purified, intact chloroplasts aqueously isolated from Wolffia arrhiza. Such chloroplasts are capable of using nitrite but not nitrate as an electron acceptor during light-stimulated electron transport in the absence of additional cytoplasmic components. When nitrite acts as an electron acceptor under these conditions, on the average 1.5 moles of oxygen are evolved per mole of nitrite reduced by the chloroplasts, indicating a probable reduction of nitrite to ammonia. Chloroplasts ruptured by osmotic shock fail to reduce nitrite in the absence of additional components.     

Characterization of the gill symbiont of Thyasira flexuosa (Thyasiridae: Bivalvia) by use of polymerase chain reaction and 16S rRNA sequence analysis.

Comparative molecular sequence (16S rRNA) analysis methods were used to identify and characterize the symbionts of Thyasira flexuosa independently of pure culture techniques and to compare these symbionts with the previously reported putative symbiont isolate, Thiobacillus thyasiris TG-2 (A. P. Wood and D. P. Kelly, Arch. Microbiol. 152:160-166, 1989). Polymerase chain reaction amplification using 16S rRNA primers specific for eubacteria was used to amplify a single unique sequence from the gill tissue of T. flexuosa. This sequence is phylogenetically most closely related to the 16S rRNA genes of known symbionts of lucinid clams and is distinct from those determined for strain TG-2 and other known bacteria. Strain TG-2 most closely resembles a free-living, chemolithoautotrophic bacterium known to be associated with the surfaces of thiotrophic bivalve shells, suggesting that this strain is a contaminant and not the authentic intracellular symbiont of T. flexuosa.     

Transformation of phosphorus and relevant intracellular compounds by a phosphorus-accumulating enrichment culture in the presence of both the electron acceptor and electron donor

This study was conducted to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms relative to the transformations of relevant intracellular compounds as well as phosphorus and nitrate for enhanced biological phosphorus removal under different combinations of electron acceptor (oxygen or nitrate) and electron donor (acetate). Under anoxic conditions, the amount of polyhydroxybutyrate (PHB) produced per acetate taken up considerably increased with the increasing amount of nitrate reduced whereas the amounts of nitrate reduced and phosphorus released per acetate taken up remained almost constant. However, glycogen utilization occurred during PHB production and then was again observed in response to the initial supplementation of acetate after glycogen accumulation was transiently observed during anoxic phosphorus uptake using nitrate as an electron acceptor. On the other hand, under subsequent aerobic conditions, the additional supplementation of acetate again caused aerobic phosphorus release and PHB production, which showed that PHB production was associated with polyphosphate cleavage regardless of electron acceptor conditions. In contrast to anoxic conditions, glycogen accumulation was observed during PHB production. Based on these observations, the preliminary model for the metabolic behavior of denitrifying phosphate-accumulating organisms was proposed and could well account for the complex transformations of PHB and glycogen together with phosphorus release in the presence of acetate under different electron acceptors.     

Lucinidae/sulfur-oxidizing bacteria: ancestral heritage or opportunistic association? Further insights from the Bohol Sea (the Philippines)

The first studies of the 16S rRNA gene diversity of the bacterial symbionts found in lucinid clams did not clarify how symbiotic associations had evolved in this group. Indeed, although species-specific associations deriving from a putative ancestral symbiotic association have been described (coevolution scenario), associations between the same bacterial species and various host species (opportunistic scenario) have also been described. Here, we carried out a comparative molecular analysis of hosts, based on 18S and 28S rRNA gene sequences, and of symbionts, based on 16S rRNA gene sequences, to determine as to which evolutionary scenario led to modern lucinid/symbiont associations. For all sequences analyzed, we found only three bacterial symbiont species, two of which are harbored by lucinids colonizing mangrove swamps. The last symbiont is the most common and was found to be independent of biotope or depth. Another interesting feature is the similarity of ctenidial organization of lucinids from the Philippines to those described previously, with the exception that two bacterial morphotypes were observed in two different species (Gloverina rectangularis and Myrtea flabelliformis). Thus, there is apparently no specific association between Lucinidae and their symbionts, the association taking place according to which bacterial species is present in the environment.     

Anaerobic respiratory growth of Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi with trimethylamine N-oxide, nitrate and fumarate: ecological implications

Two symbiotic species, Photobacterium leiognathi and Vibrio fischeri, and one non-symbiotic species, Vibrio harveyi, of the Vibrionaceae were tested for their ability to grow by anaerobic respiration on various electron acceptors, including trimethylamine N-oxide (TMAO) and dimethylsulphoxide (DMSO), compounds common in the marine environment. Each species was able to grow anaerobically with TMAO, nitrate or fumarate, but not with DMSO, as an electron acceptor. Cell growth under microaerophilic growth conditions resulted in elevated levels of TMAO reductase, nitrate reductase and fumarate reductase activity in each strain, whereas growth in the presence of the respective substrate for each enzyme further elevated enzyme activity. TMAO reductase specific activity was the highest of all the reductases. Interestingly, the bacteria-colonized light organs from the two squids, Euprymna scolopes and Euprymna morsei, and the light organ of the ponyfish, Leiognathus equus, also had high levels of TMAO reductase enzyme activity, in contrast to non-symbiotic tissues. The ability of these bacterial symbionts to support cell growth by respiration with TMAO may conceivably eliminate the competition for oxygen needed for both bioluminescence and metabolism.     

Anaerobic biodegradation of ethylthionocarbamate by the mixed bacteria under various electron acceptor conditions

Biodegradation behavior and kinetics of ethylthionocarbamate under nitrate, sulfate and ferric reducing conditions by mixed cultures enriched from the anaerobic digester sludge was investigated. The results showed that ethylthionocarbamate could be degraded independently by the mixed cultures coupled to nitrate, sulfate, and ferric reduction, and meanwhile, nitrite, sulfide, and ferrous were accumulated as a result of nitrate, sulfate and ferric reduction, respectively. Ferric was a more favorable terminal electron acceptor compared to nitrate and sulfate. The order of the electron acceptors with decreasing biodegradation rates of the ethylthionocarbamate was: ferric>nitrate>sulfate, and the corresponding maximum biodegradation rate was 7.240, 6.267, and 4.602 mg/(L·d), respectively. The anaerobic biodegradation of ethylthionocarbamate under various electron acceptor conditions can be accurately described by first order exponential decay kinetics.     

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.     

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.     

Biochemistry of nitrate respiration in Pseudomonas stutzeri. I. Aerobic and nitrate respiration routes of carbohydrate catabolism

Spangler, W. J. (Oregon State University, Corvallis), and C. M. Gilmour. Biochemistry of nitrate respiration in Pseudomonas stutzeri. I. Aerobic and nitrate respiration routes of carbohydrate catabolism. J. Bacteriol. 91:245-250. 1966.-The metabolic pathways of glucose catabolism were studied in Pseudomonas stutzeri under aerobic conditions and under conditions of nitrate respiration. Studies on both glucose and gluconate catabolism, by the radiorespirometric method, indicated that these substrates are degraded in the same manner, i.e., the Entner-Doudoroff and pentose phosphate pathways. There appeared to be no major shift in primary metabolic pathways when nitrate was used as the terminal hydrogen acceptor in nitrate respiration as opposed to aerobic respiration with free molecular oxygen. It was shown that glucose is not degraded to any appreciable extent under anaerobic conditions in the absence of nitrate. Tentative evidence suggests that the tricarboxylic acid cycle functions under both conditions of oxygen relationships and that the rate of carbon oxidation via the tricarboxylic acid cycle is slower with nitrate respiration than under aerobic conditions.     

Gill filament differentiation and experimental colonization by symbiotic bacteria in aposymbiotic juveniles of Codakia orbicularis (Bivalvia: Lucinidae)

Summary

Codakia orbicularis is a tropical lucinid harboring gill endosymbionts which are environmentally transmitted from a free living-symbiont form to the new host generation after metamorphosis. Structural changes occurring in the cellular organization from incomplete gill filaments in young aposymbiotic juveniles to full differentiated gill filaments containing bacterial endosymbionts in reared symbiotic juveniles, were analyzed for juveniles from 250 μm to 2 μm shell-length. Aposymbiotic juveniles possess differentiated gill filaments with ciliated, intermediary, and lateral zones similar to those described in wild juveniles, except for the bacteriocytes which are lacking. Granule cells, which progressively differentiate during the morphogenesis of the gill filament, do not appear as a consequence of symbiosis. Experimental colonization of aposymbiotic juveniles by the free-living symbiont form has been obtained through the addition of unsterilized sand collected from the natural habitat of C. orbicularis. Two days after exposure to crude sand, symbiosis-competent bacteria enter by endocytosis at the apical pole of undifferentiated cells which progressively differentiate into classical bacteriocytes similar to those found in the adult gill filaments. Undifferentiated cells of aposymbiotic gill filaments remain receptive to bacteria several months after metamorphosis, and become bacteriocytes when aposymbiotic juveniles get contact with the symbiont free-living form. Therefore, the environmental transmission of symbionts does not appear to be restrained to a defined period of time during post-larval development in C. orbicularis.     

Inhibition of respiration and nitrate assimilation enhances photohydrogen evolution under low oxygen concentrations in Synechocystis sp. PCC 6803

In cyanobacterial membranes photosynthetic light reaction and respiration are intertwined. It was shown that the single hydrogenase of Synechocystis sp. PCC 6803 is connected to the light reaction. We conducted measurements of hydrogenase activity, fermentative hydrogen evolution and photohydrogen production of deletion mutants of respiratory electron transport complexes. All single, double and triple mutants of the three terminal respiratory oxidases and the ndhB-mutant without a functional complex I were studied. After activating the hydrogenase by applying anaerobic conditions in the dark hydrogen production was measured at the onset of light. Under these conditions respiratory capacity and amount of photohydrogen produced were found to be inversely correlated. Especially the absence of the quinol oxidase induced an increased hydrogenase activity and an increased production of hydrogen in the light compared to wild type cells. Our results support that the hydrogenase as well as the quinol oxidase function as electron valves under low oxygen concentrations. When the activities of photosystem II and I (PSII and PSI) are not in equilibrium or in case that the light reaction is working at a higher pace than the dark reaction, the hydrogenase is necessary to prevent an acceptor side limitation of PSI, and the quinol oxidase to prevent an overreduction of the plastoquinone pool (acceptor side of PSII). Besides oxygen, nitrate assimilation was found to be an important electron sink. Inhibition of nitrate reductase resulted in an increased fermentative hydrogen production as well as higher amounts of photohydrogen.     

Continuous rhamnolipid production using denitrifying Pseudomonas aeruginosa cells in hollow‐fiber bioreactor

Rhamnolipids are high‐value effective biosurfactants produced by Pseudomonas aeruginosa. Large‐scale production of rhamnolipids is still challenging especially under free‐cell aerobic conditions in which the highly foaming nature of the culture broth reduces the productivity of the process. Immobilized systems relying on oxygen as electron acceptor have been previously investigated but oxygen transfer limitation presents difficulties for continuous rhamnolipid production. A coupled system using immobilized cells and nitrate instead of oxygen as electron acceptor taking advantage of the ability of P. aeruginosa to perform nitrate respiration was evaluated. This denitrification‐based immobilized approach based on a hollow‐fiber setup eliminated the transfer limitation problems and was found suitable for continuous rhamnolipid production in a period longer than 1,500 h. It completely eliminated the foaming difficulties related to aerobic systems with a comparable specific productivity of 0.017 g/(g dry cells)‐h and allowed easy recovery of rhamnolipids from the cell‐free medium. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346–351, 2013