Photosynthetic Bacterium Rhodopseudomonas Palustris Research Articles

Overexpression of ATP Synthase Subunit Beta (Atp2) Confers Enhanced Blast Disease Resistance in Transgenic Rice.

Previous research has shown that the pathogenicity and appressorium development of Magnaporthe oryzae can be inhibited by the ATP synthase subunit beta (Atp2) present in the photosynthetic bacterium Rhodopseudomonas palustris. In the present study, transgenic plants overexpressing the ATP2 gene were generated via genetic transformation in the Zhonghua11 (ZH11) genetic background. We compared the blast resistance and immune response of ATP2-overexpressing lines and wild-type plants. The expression of the Atp2 protein and the physiology, biochemistry, and growth traits of the mutant plants were also examined. The results showed that, compared with the wild-type plant ZH11, transgenic rice plants heterologously expressing ATP2 had no significant defects in agronomic traits, but the disease lesions caused by the rice blast fungus were significantly reduced. When infected by the rice blast fungus, the transgenic rice plants exhibited stronger antioxidant enzyme activity and a greater ratio of chlorophyll a to chlorophyll b. Furthermore, the immune response was triggered stronger in transgenic rice, especially the increase in reactive oxygen species (ROS), was more strongly triggered in plants. In summary, the expression of ATP2 as an antifungal protein in rice could improve the ability of rice to resist rice blast.

Mode of autophosphorylation in bacteriophytochromes RpBphP2 and RpBphP3.

Phytochromes are red-light photoreceptors that regulate a wide range of physiological processes in plants, fungi and bacteria. Canonical bacteriophytochromes are photosensory histidine kinases that undergo light-dependent autophosphorylation, thereby regulating cellular responses to red light via two-component signaling pathways. However, the molecular mechanism of kinase activation remains elusive for bacteriophytochromes. In particular, the directionality of autophosphorylation is still an open question in these dimeric photoreceptor kinases. In this work, we perform histidine kinase assays on two tandem bacteriophytochromes RpBphP2 and RpBphP3 from the photosynthetic bacterium Rhodopseudomonas palustris. By examining the kinase activities of full-length bacteriophytochromes and two loss-of-function mutants under different light conditions, we demonstrate that RpBphP2 and RpBphP3 undergo light-dependent trans-phosphorylation between protomers in both homodimeric and heterodimeric forms. We have further determined the crystal structure of the histidine kinase domains of RpBphP2 at 3.19Å resolution. Based on structural comparisons and homology modeling, we also present a model to account for the actions of trans-autophosphorylation in bacteriophytochromes.

Characterization of CdSe QDs biosynthesized by a recombinant Rhodopseudomonas palustris

This study proposed a novel approach for the biosynthesis of CdSe quantum dots (QDs) by a recombinant photosynthetic bacteria Rhodopseudomonas palustris with metallothionein expression. The biosynthesis mechanisms and physico-chemical properties of the CdSe QDs were analyzed by various techniques, and its potential application was also investigated. The results showed that the biosynthesized CdSe QDs presented green fluorescence with maximum excitation and emission wavelengths at 460 and 530 nm, respectively. The quantum yield of the biosynthesized CdSe QDs was found out to be 3.85%. They were well-dispersed spherical particles with an average size of 5.37 ± 0.06 nm. Their crystal structure was cubic with the d spacings of 3.1 Å and 2.5 Å, corresponded to the planes of (111) and (220), respectively. The biosynthesis process of CdSe QDs took place in the cytoplasm and may involve some proteins, peptides, lipids or other organic substances covering the surface of CdSe QDs through several specific functional groups, such as hydroxyl or sulfhydryl groups to maintain their stability. The purified CdSe QDs had good biocompatibility and great potential for cellular imaging application. It is concluded that the process may provide a more cost-effective and eco-friendly method not only to recover harmful cadmium in the environment but also to synthesize simultaneously a clean product for various industrial applications.

Combining the microbial agent Rhodopseudomonas palustris strain PSB-06 with fungicides for controlling rice blast

The rice blast disease caused by Magnaporthe oryzae threatens global rice production yields. Tricyclazole and isoprothiolane are widely used fungicides with high activity against rice blast, and our previous study indicated the photosynthetic bacterium Rhodopseudomonas palustris PSB-06 significantly antagonizes rice blast. However the effect of combining these two chemical fungicides with PSB-06 on rice blast control is unclear. Here we test the control effect of photosynthetic bacteria PSB-06 combined with isoprothiolane and tricyclazole on rice blast. The growth of PSB-06 was unaffected by up to 1.25 mg/L of tricyclazole and 0.3 mg/L of isoprothiolane in the photosynthetic medium, indicated the two fungicides have no inhibition on PSB-06. The control efficiency in the field test reached 76.06% when PSB-06 was combined with isoprothiolane. This value was significantly higher than the individual efficiency of PSB-06 (67.99%) and tricyclazole (65.46%) and the combined control efficiency (72.20%) of those two antifungal agents. Our current findings highlighted the potential of combining R. palustris strain PSB-06 with isoprothiolane to control rice blast, providing environmental protection and reducing the use of fungicides.

Extracellular electron uptake for CO2 fixation by Rhodopseudomonas palustris during electro-cultivation in darkness

As a vital part of the global carbon cycle, photosynthesis helps in fixing CO2 to produce diverse biomass. However, over-reliance on optical density results in inadequate photosynthesis under limited light sources. The coupling of extracellular respiration and photosynthetic chain via the quinone pool provides a possibility for electrically driven photosynthesis in darkness, which is not well understood. In this study, CO2 fixation of photosynthetic bacteria Rhodopseudomonas palustris was enhanced in the dark via extracellular electron uptake from the electrode at −0.4 V. The copy number of R. palustris increased by 35 folds during 28 days of operation, accompanied by the increase of ATP content, NADH/NAD+, and NADPH/NADP+ of cells. Especially, the activity of Rubisco, the key enzyme of the Calvin cycle, increased by 28 % during electro-cultivation. Accordingly, the electrochemical activity of R. palustris was found to increase, which might be attributed to the structural modification of protein-like substances due to the enhanced proton-coupled electron transfer (PCET) process in electro-cultivation, which was further confirmed by in situ Fourier transform infrared spectroscopy and kinetic isotope effect tests. This study indicated that extracellular respiration could be electrostimulated via PCET to maintain photosynthesis in R. palustris in the dark.

Proteomic analysis of the electron uptake pathway of Rhodopseudomonas palustris CGA009 under different cathodic potentials

Microbial electrosynthesis can convert greenhouse gas CO2 into value-added chemicals at room temperature and mild conditions, and the efficiency of its reaction is closely dependent on the electrode potential and electron transport chain-related functional proteins. In this study, the photosynthetic bacterium Rhodopseudomonas palustris CGA009 was used to explore CO2 bioreduction behavior at different potentials (−0.2 V, −0.5 V, −0.8 V). Label-free quantitative proteomics was used to analyze the mechanism of microbial electron transfer. The results showed that the cathodic potential could positively affect the NAD(P)-binding domain-containing protein of R. palustris CGA009. Ubiquinone and other terpenoid-quinone biosynthesis pathways that are mainly responsible for NAD(P)-binding domain-containing proteins also showed an upregulating trend in the KEGG analysis. The electrons are transferred to the photosynthetic chain through the B800-850 complex to collect light energy, and then the photosynthetic reaction center uses this energy to generate a proton gradient for transmembrane transport, allowing R. palustris CGA009 to use electrons for the fixation process of CO2. Our results enlighten the role of proteins in electron transfer chain of microbial electrosynthesis and provide an insight into how to regulate the microbial process to produce value-added chemicals.

A Red Fluorescent Protein Reporter System Developed for Measuring Gene Expression in Photosynthetic Bacteria under Anaerobic Conditions.

The photosynthetic bacterium Rhodopseudomonas palustris converts nitrogen gas (N2) to fertilizer ammonia (NH3) and also produces clean energy hydrogen gas (H2) from protons (H+) when it is grown anaerobically in nitrogen fixing medium with illumination, a condition that promotes the expression of active nitrogenase. Compared with quantitative real-time PCR (qRT-PCR) and the lacZ reporter system, two methods commonly used for in vivo study of nitrogenase regulation in photosynthetic bacteria, the fluorescent protein reporter system has advantages in terms of its simplicity and sensitivity. However, little is known concerning if the fluorescent protein reporter system can be used in bacterial cells that need to grow anaerobically. Here, we developed an RFP-based method to measure the nitrogenase gene expression in photosynthetic bacteria grown anaerobically. This method was able to determine the levels of both the genome-based and the plasmid-based nitrogenase expression under anaerobic conditions, providing a better method for in vivo study of gene expression affected by oxygen. The RFP reporter system developed here will promote a better understanding of the molecular mechanism of nitrogenase regulation and will be used on other genes of interest in a wider range of anaerobic bacteria.

English

This study investigated the use of effective microorganisms (EM) to enhance cost-effective biogas purification at household-level application. It involved experimental setups for biogas purification in two different runs: 4 L activated EM and 1 L dormant EM by bubbling biogas through EM purification units. Biogas composition was analyzed using an industry-standard biogas analyzer. The results indicated that EM has the potential for biogas purification through a biological process, to remove H2S and CO2 by involving photosynthetic bacteria (Rhodopseudomonas palustris) in the presence of light. The raw biogas average composition was 62.2% CH4, 37.4% CO2 and 1359.3 ppm H2S. The 4L activated EM batch solution purified the raw biogas to 80.2% CH4, 19.5% CO2 and 786.1 ppm H2S. The corresponding purification efficiencies are 60% for CO2 and 49% for H2S. Purified biogas could be used by the household to cook for 1.82 h (85%) while raw biogas could cover only 55%. The designed biogas purification system can cost only Tshs 91,010/= and purify about 15,000 L of biogas at EM cost of Tshs 9,100/month.   Key words: Biogas composition, photosynthetic bacteria, anaerobic digestion, biochemical processes, Rhodopseudomonas palustris, biological desulphurization.

GroEL protein from the potential biocontrol agent Rhodopseudomonas palustris enhances resistance to rice blast disease.

GroEL, which is a chaperone, plays a key role in maintaining protein homeostasis and, among other functions, serves to prevent protein misfolding and aggregation. In addition, the GroEL protein also has a significant effect on enhancing plant resistance and inhibiting plant diseases. However, the function of the GroEL protein in the inhibition of rice blast remains unknown. Field experiment results show that photosynthetic bacteria PSB-06 have a good control effect on Magnaporthe oryzae. PSB-06 also can promote rice growth and enhance stress resistance. A GroEL protein which was separated and purified from photosynthetic bacteria had a significant antagonistic effect on appressorial formation and pathogenicity of Magnaporthe oryzae, meanwhile transcriptional analysis demonstrated that the GroEL protein could improve the expression of defense gene of rice. Our results show that the photosynthetic bacteria Rhodopseudomonas palustris significantly controls rice blast disease. Its action involves an extracellular GroEL protein, which inhibits appressoria formation, antagonizes the pathogenicity of Magnaporthe oryzae and promotes a host defense response. The research results provide evidence of the potential of this photosynthetic bacterium as a biocontrol agent at least for rice blast control. © 2021 Society of Chemical Industry.

The Photosynthetic Bacterium Rhodopseudomonas palustris Strain PS3 Exerts Plant Growth-Promoting Effects by Stimulating Nitrogen Uptake and Elevating Auxin Levels in Expanding Leaves.

Rhodopseudomonas palustris strain PS3, a phototrophic bacterium, was originally isolated from a paddy field located in Taipei city, Taiwan, and showed positive effects on the growth of leafy vegetables. The aim of this study was to clarify the mechanism of the beneficial effects exerted by PS3 on plants. An ineffective R. palustris strain, YSC3, isolated from a paddy field located in Yilan County, was used as the negative control for comparative analyses. We cultivated non-heading Chinese cabbage (Brassica rapa var. chinensis) in 1/2 strength Hoagland hydroponic solution, in which nitrate is the main nitrogen source. We evaluated various plant physiological responses to inoculation with different bacterial inoculants. The N use efficiency (NUE) of PS3-inoculated plants was dramatically higher than that of YSC3-inoculated plants. The nitrate uptake efficiency (NUpE) was significantly elevated in plants treated with PS3; however, no excess nitrate accumulation was observed in leaves. We also noticed that the endogenous indole-3-acetic acid (IAA) levels as well as the cell division rate in the leaves of PS3-inoculated plants were significantly higher than those in the leaves of YSC3-inoculated plants. We examined the bacterial transcription of some genes during root colonization, and found that the expression level of IAA synthesis related gene MAO was almost the same between these two strains. It suggests that the elevated endogenous IAA in the PS3-inoculated plants was not directly derived from the exogenous IAA produced by this bacterium. Taken together, we deduced that PS3 inoculation could promote plant growth by enhancing nitrate uptake and stimulating the accumulation of endogenous auxin in young expanding leaves to increase the proliferation of leaf cells during leaf development.

Enhancement of biodiesel-promising microalgae Chlorella pyrenoidosa growth using stimulants in municipal sewage

Cultivation of high-lipid microalgae using municipal sewage can both treat polluted water and gain biodiesel. Rapid cultivation of microalgae is crucial for the entire process. In this study, four stimulants (phytohormone 2,4-d; Fe3+; photosynthetic bacteria—Rhodopseudomonas palustris; CO2) were firstly chosen to investigate their effects on microalgae Chlorella pyrenoidosa cultivation in municipal sewage. Meanwhile, orthogonal experiments were designed to compare the enhancing effects on the growth of microalgae C. pyrenoidosa and lipid accumulation by different combinations of stimulants, which were also conducted for outdoor culture. The optimum stimulant combination was 1.0 mg/L 2,4-d; 10−7 mol/L Fe3+; 4.5 mL (109 cells/mL) photosynthetic bacteria; and 8% CO2. Among the four kinds of stimulants, phytohormone 2,4-d and photosynthetic bacteria played more important roles on the microalgae growth and lipid accumulation. The optimum combination of stimulants could also promote the microalgae biomass and lipid yield during outdoor cultivation. The maximum OD681 of microalgae was 37.9% higher than that of the control group without addition of stimulants. The obtained microalgae dry weight and lipid production were 0.3 and 0.13 g /L, which were 80.0% and 85.7% higher than that of the control group, respectively. The promotion of microalgae photosynthetic rate was the main reason for the stimulant-enhanced microalgae cultivation. This study exerts positive effects on the development of microalgae biodiesel.

Optimization of three anoxygenic photosynthetic bacteria as feed to enhance growth, survival, and water quality in fairy shrimp (Streptocephalus sirindhornae) cultivation

The fairy shrimp Streptocephalus sirindhornae has been commercially produced as a potential live feed to replace the use of brine shrimp in freshwater aquaculture. To determine an optimal diet for these fairy shrimp, the use of highly nutritious microorganisms that are relatively simple and cost effective to cultivate should be explored. In this study, the anoxygenic photosynthetic bacteria Rhodobacter sphaeroides, Rhodopseudomonas palustris, and Rhodopseudomonas faecalis PA2 were assessed for fairy shrimp cultivation, and the effect of dried bacterial biomass is reported. Fairy shrimp fed R. faecalis PA2 showed the highest survival and growth performance in both larval and adult experiments. At the adult stage, the survival and growth of fairy shrimp fed R. faecalis PA2 were higher than those of the control, whereas those fed R. sphaeroides complete died at 12 days of age. Dissolved oxygen (DO), ammonia, nitrite, and nitrate concentrations in the treatments containing photosynthetic bacteria were acceptable. The effects of air-dried (without sunlight) R. faecalis PA2 (AD) and freeze-dried R. faecalis PA2 (FD) on fairy shrimp were investigated and compared with those of commercial dried Spirulina (SP), a bacterial suspension of R. faecalis PA2 (BS), and a control treatment. The maximum survival rate and growth performance were observed in fairy shrimp fed BS; meanwhile, those fed AD, FD, and the control had similar results, which included slightly lower survival than those fed the BS. Surprisingly, the SP treatment group had the lowest survival rate and DO concentration as well as the highest ammonia concentration. Analyses of proximate composition showed that the nutritional values of AD and FD, particularly in terms of their protein and carbohydrate contents, were favorable for aquatic feeds. Our results suggest that R. faecalis PA2 may be used as a new feed for fairy shrimp to enhance growth and survival with no effect on water quality. Although the fresh form was superior, the dry form is also a satisfactory option if storage time is a concern.

Solvent-free chlorophyll spectrometry in unicellular algal research

Scanning spectrophotometers equipped with integrating spheres are now readily available commercially. This study explores the possibility of largely escaping from solvent-based determinations of chlorophylls and bacteriochlorophylls using the in vivo absorption spectra to deduce the chlorophyll/bacteriochlorophyll (Chl/BChl) contents of unicellular photosynthetic organisms. Furthermore, we show that scans from ordinary dual beam spectrometers can also be used to estimate the Chl/BChls of unicells in vivo despite light scattering. Spectra of unicellular algae using integrating sphere spectroscopy can measure the absorbance (A) of turbid cell suspensions and hence the in vivo pigment absorption properties of photosynthetic organisms. These results were compared with those obtained using conventional dual beam spectrophotometry scans on turbid cell suspensions and the in solvent spectra of the photosynthetic pigments. The common unicellular green alga, Chlorella sp., is used as an example of an oxygenic photo-organism with Chl a + b, and comparisons were made to other unicellular algae: a cyanobacterium (Synechococcus), a diatom (Chaetoceros) and Acaryochloris. The anoxygenic photosynthetic bacterium Rhodopseudomonas palustris (BChl a) was included in the study. Empirical algorithms are presented for the solvent-free estimation of Chls a, a + b, a + c1c2, d + a and BChl a for the organisms in the study.

Integrated control of potato late blight with a combination of the photosynthetic bacterium Rhodopseudomonas palustris strain GJ-22 and fungicides

Late blight caused by the oomycete Phytophthora infestans is the major yield-threatening disease in potato production. An isolate of the photosynthetic bacterium Rhodopseudomonas palustris, GJ-22, was previously evaluated for its ability to promote the growth and enhance the immune response of solanaceous plants. In the present study, GJ-22 was shown to significantly repress disease symptom development caused by P. infestans in potato tubers. Curzate (cymoxanil plus mancozeb), a widely used fungicide with high activity against P. infestans, was selected for use in combination with GJ-22. The sensitivity of GJ-22 to the fungicides cymoxanil and mancozeb was tested. The growth of GJ-22 was unaffected by up to 100 mg l−1 cymoxanil or 50 mg l−1 mancozeb in photosynthetic medium. In greenhouse and field experiments, the combined application of GJ-22 and curzate resulted in better disease control than the use of either agent alone. In addition, treatment with 1 × 107 CFU ml−1 GJ-22 plus 700 mg l−1 curzate showed synergistic effects in the field trial. Our results highlight the potential to control potato late blight with the combination of R. palustris strain GJ-22 and curzate, providing environmentally friendly protection with a reduced level of fungicide application.

Photoheterotrophy of photosynthetic bacteria ( Rhodopseudomonas palustris ) growing on oil palm and soybean cooking oils

Abstract Cooking oil wastes are environmentally damaging and costly to deal with. Photoheterotrophic growth of Rhodopseudomonas palustris (photosynthetic bacteria) (CGA 009) was used to decompose different types of cooking oil (palm and soybean oils; cooked and uncooked palm oil) under anaerobic and microaerobic conditions. Photosynthesis was measured in R. palustris using PAM technology (Pulse Amplitude Modulation Fluorometry): Optimum irradiance ≈ 284 μ mol photon m−2 s−1 and maximum photosynthetic electron transport rate (ETR) ≈ 341 μ mol e − g−1 BChl a s−1. R. palustris grew photoheterotrophically on both cooking oils as the sole carbon source grown anaerobically and microaerobically in PM media with a NH3-nitrogen source. R. palustris grew well on used palm oil. R. palustris produces small volumes of hydrogen gas from biodegradation of oil but only under nitrogen fixing conditions (anaerobic: no ammonia or organic nitrogen). N-fixing cells grew very slowly. Cooking oil waste residues are also produced at the production stage (Palm Oil Milling Effluent, POME) and in recycling them for reuse or to produce biodiesel. Hence, biodegradation of cooking oil wastes is an important issue at the production, domestic consumption and recycling stages of their use. A two-stage digestion using an anaerobic yeast first stage, followed by a R. palustris digestion was feasible. Two Line Capsule: The photosynthetic bacterium R. palustris grows photoheterotrophically on Palm Oil and Soybean Oil. Photosynthetic rates were measured using PAM technology.

Increase of Salt Tolerance in Carbon-Starved Cells of Rhodopseudomonas palustris Depending on Photosynthesis or Respiration.

Bacteria in natural environments are frequently exposed to nutrient starvation and survive against environmental stresses under non-growing conditions. In order to determine the energetic influence on survivability during starvation, changes in salt tolerance were investigated using the purple photosynthetic bacterium Rhodopseudomonas palustris after carbon starvation under photosynthetic conditions in comparison with anaerobic and aerobic dark conditions. Tolerance to a treatment with high concentration of salt (2.5 M NaCl for 1 h) was largely increased after starvation under anaerobically light and aerobically dark conditions. The starved cells under the conditions of photosynthesis or aerobic respiration contained high levels of cellular ATP, but starvation under the anaerobic dark conditions resulted in a decrease of cellular ATP contents. To observe the large increase of the salt tolerance, incubation of starved cells for more than 18 h under illumination was needed. These results suggest that the ATP-dependent rearrangement of cells induced salt tolerance.

Synthesis of Carotenoids of Industrial Interest in the Photosynthetic Bacterium Rhodopseudomonas palustris : Bioengineering and Growth Conditions.

Rhodopseudomonas palustris is a purple photosynthetic bacterium that accumulates in the inner membrane the photosynthetic pigment spirilloxanthin, formed from lycopene. Here, we describe the procedures used to successfully engineer Rps. palustris strains to reroute the production of lycopene toward the synthesis of ß-carotene or canthaxanthin. The crtCD genes specifically involved in spirilloxanthin were replaced by crtY and crtW genes from Bradyrhizobium ORS278 to synthesize ß-carotene and (or) canthaxanthin, two pigments of industrial interest. Since the synthesis of canthaxanthin depends on the presence of oxygen, the procedure to optimize their production is also proposed. By modulating the light and oxygen during the growth process, a single species of photosynthetic bacteria, with an efficient growth rate, produces various carotenoids of economical interest.

Apoprotein heterogeneity increases spectral disorder and a step-wise modification of the B850 fluorescence peak position

It has already been established that the quaternary structure of the main light-harvesting complex (LH2) from the photosynthetic bacterium Rhodopseudomonas palustris is a nonameric ‘ring’ of PucAB heterodimers and under low-light culturing conditions an increased diversity of PucB synthesis occurs. In this work, single molecule fluorescence emission studies show that different classes of LH2 ‘rings’ are present in “low-light” adapted cells and that an unknown chaperon process creates multiple sub-types of ‘rings’ with more conformational sub-states and configurations. This increase in spectral disorder significantly augments the cross-section for photon absorption and subsequent energy flow to the reaction centre trap when photon availability is a limiting factor. This work highlights yet another variant used by phototrophs to gather energy for cellular development.

Single stage hydrogen production from cellulose through photo-fermentation by a co-culture of Cellulomonas fimi and Rhodopseudomonas palustris

Biohydrogen production from cellulose by a bacterial co-culture is a potentially promising approach for producing bioenergy from a low cost substrate. The use of a cellulolytic bacterium, Cellulomonas fimi, permits cellulose conversion and the in situ production of substrate for growth and hydrogen production by the photosynthetic bacterium Rhodopseudomonas palustris. Response surface methodology (RSM) with a Box-Behnken design (BBD) was used to examine variations in the key parameters: substrate (cellulose) concentration, yeast extract concentration and the microorganism ratio (Rps. palustris/C. fimi). For the co-culture of R. palustris and C. fimi the highest hydrogen production (44 mmol H2/L) was achieved at the highest substrate concentration (5 g/L); however, the highest hydrogen yield (3.84 mol H2/mol glucose equivalent) was observed at the lowest cellulose concentration and highest microorganism ratio. High COD removal efficiencies, over 70%, were achieved over a wide range of conditions and were positively affected by the concentration of yeast extract.

Mutation design of a thermophilic Rubisco based on three-dimensional structure enhances its activity at ambient temperature.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc.