The sensing, response and subsequent acclimation of plants to suboptimal and adverse conditions is of pivotal importance to productivity and crop yield. Natural fluctuations in environmental growth conditions have the potential to strongly alter growth rate and adversely affect fitness of photosynthetic organisms. However, plants and algae have developed elaborate acclimation mechanisms that fine tune the metabolism of the organism to this altered environment, enabling cells to adjust and optimize growth capacity upon establishing a new homeostasis. My Ph.D. research was focused on identification of novel genes and proteins that are involved in the signal transduction pathway involved in these acclimation mechanisms to fluctuations in carbon-dioxide level in the unicellular green alga Chlamydomonas reinhardtii.
Approximately 50% of the world’s photosynthesis occurs in the aquatic environment by organisms that employ a carbon concentrating mechanism (CCM). Aquatic photosynthetic organisms have evolved different forms of CCMs to aid the enzyme Rubisco in capturing CO2 from the surrounding environment. One aspect of all CCMs is the critical roles played by various extracellular and intracellular carbonic anhydrases (CAs). CAs are of four sub types namely alpha, beta, gamma and epsilon. My PhD research was focused on identification of some novel carbonic anhydrase genes in C. reinhardtii, a green alga with a well studied CO2 concentrating mechanism (CCM). At the time of my PhD study, five carbonic anhydrases were known to exist in C. reinhardtii. I have identified two novel genes encoding beta type CA (CAH6 and CAH8) and two gamma CA like genes (GLP1 and GLP2. Two of these proteins, Cah6 (chloroplastic) and GLP1 (cytoplasmic) and a known thylakoid alpha CA (CAH3) protein were overexpressed as recombinant MBP (maltose binding protein)-fusion proteins to assay enzyme activities. I demonstrated that the recombinant CAH6 and CAH3 fusion proteins are enzymatically active but GLP1 is not. The purified recombinant CAH6 and CAH3 proteins were used to raise antibodies for immunolocalization and biochemical studies. RNA interference, a powerful gene silencing tool, was employed, along with traditional molecular biological methods like Northern and Western blotting, to study the functional role of CAH6 in the CCM and photosynthesis.
My post-doctoral research was focused on identification of novel genes and proteins that are involved in the signal transduction pathway involved in these acclimation mechanisms to fluctuations in irradiance in the unicellular green alga Chlamydomonas reinhardtii. My postdoctoral research project specifically involved identifying and characterizing novel genes that define the chlorophyll antenna size in the model green alga C. reinhardtii employing biochemical and molecular approaches. Up to 600 chlorophyll (Chl) molecules associated with the two photosystems in the chloroplast of C. reinhardtii, are needed to increase the photon absorption cross-section of the photosynthetic apparatus. This large Chl antenna size affords the cells a competitive advantage in the wild, where sunlight is often limiting. However, only about 130 Chl molecules are absolutely needed for the proper assembly and function of the photosystems. The Chl antenna size of the photosynthetic apparatus in chloroplasts is defined genetically by the nucleus via an unknown control mechanism. Specifically, there are unknown nuclear genes that regulate the development and define the size of the Chl antenna in all photosynthetic organisms.
tla1, (truncated light harvesting chlorophyll antenna size) a chlorophyll deficient insertional mutant with a smaller Chl antenna size compared to that of the wild type was identified upon screening of an insertional mutant library. TAIL/Inverse PCR analysis revealed that in the mutant the 5’ UTR and the promoter of a novel TLA1 gene was misplaced from its original location to a location upstream of the inserted vector that was used for insertional mutagenesis. RT-PCR and 5’RACE analysis using the cDNA of the mutant and the wild type revealed that TLA1 transcript is expressed in the mutant and that the 3’end of the inserted vector was acting as the new 5’UTR and promoter in the mutant. Moreover, I identified a novel gene RDP1 that overlaps at its 3’end with the 5’end of the TLA1 gene. The TLA1 gene encodes a novel protein of 213 amino acids and shows substantial homology to proteins of unknown function in diverse eukaryotic organisms, ranging from higher plants to invertebrates and mammals. RDP1 codes for a protein with zinc RING finger domain that are common in protein involved in protein turnover and regulators of transcription and translation.
TLA1 protein was overexpressed as a His-tagged recombinant fusion protein and purified it to generate an antibody for immunolocalization studies. Western blotting experiment indicated that the TLA1 protein is drastically reduced in the mutant. Complementation of the tla1 mutant strain with the full length TLA1 gene restored the wild type phenotype. Immunolocalization studies using a TLA1 specific antibody showed that TLA1 protein is localized in the chloroplast. Since the TLA1 protein belongs to a novel yet uncharacterized protein family, predicting a broad functional role of TLA1 is difficult. Through application of computational-based bioinformatics tools I have shown that the conserved domains of TLA1 like proteins have a remote homology with the plain MPN/MOV34 domains, which are domains present in proteins involved in protein degradation, translation factors and transcription regulators like subunits of COP9 signalosome (CSN6), eukaryotic translation initiation factor (eIF3f and eIF3h) etc. I performed over-expression and down-regulation of the TLA1 gene (using RNA interference) to alter the optical properties of green algae by conferring a larger or truncated, Chl antenna size. The over-expression and down regulation of the TLA1 gene affected the concentration of some of the major photosynthetic proteins and altered the organization of the thylakoid membrane structure in the chloroplast. This research has important application in algal biotechnology for improving photosynthetic productivity and solar conversion efficiency of commercially important photosynthetic microalgae, biomass accumulation and carbon sequestration (see CV for research patent).
A. PHOTOSYNTHESIS-BASED RESEARCH PROJECT USING CHLAMYDOMONAS (A GREEN YEAST/MICRO-ALGA)
I am interested broadly in identifying and characterizing novel molecular components essential for photosynthesis and photosynthetic pigment metabolism employing forward and reverse genetics in the model experimental green micro-alga Chlamydomonas reinhardtii.
Chlamydomonas reinhardtii is a green micro algae that is haploid, easy to culture in the laboratory, can grow both photosynthetically and heterotrophically (can use acetate as the sole carbon source in the dark like a heterotroph and also can use atmospheric CO2 in the presence of light, like a photosynthetic autotroph), is amenable to both nuclear and chloroplast transformation and its genome has been sequenced. All of these traits make it an ideal, simple and elegant system to study photosynthesis. Additionally, Chlamydomonas has two different pathways to make chlorophyll, unlike angiosperms. It has a strictly light dependent chlorophyll biosynthetic pathway like all photosynthetic higher plants and a light independent chlorophyll biosynthetic pathway that can operate both under dark and light, like in some cyanobacteria and gymnosperms. Photosynthesis is the only O2 generating biochemical reaction on Earth that sustains life. Photosynthesis consists of two sets of reaction, namely the light reaction and the Calvin cycle, that take place in the chloroplast of plant cells. During the light reaction, absorbed light energy is used to extract electrons from water with generation of oxygen as a byproduct. The energy from the electron is converted to chemical energy (ATP and NADPH). This converted chemical energy is then used to assimilate CO2 into carbohydrates. Antenna of a photosystem is comprised of photosynthetic pigment molecules (chlorophyll and carotenoids) bound to LHCs (light harvesting complex proteins) and is present in the thylakoid membrane of chloroplast. It is responsible for trapping solar energy and passing it on to the reaction center for photochemistry. After absorbing the light energy, chlorophyll becomes excited to its singlet excited state and then transfers the absorbed energy to the reaction centers of photosystems, where it drives the initial charge separation reactions of photosynthesis (photochemistry). Besides photochemistry, fluorescence emission, de-excitation by thermal dissipation, and decay through triplet state are some other means by which excited chlorophylls return to ground state. Light is essential for photosynthesis. When plants receive more light than they can utilize, the lifetime of singlet excited chlorophyll extends and the chance of returning to ground state through triplet state chlorophyll is increased. This pathway can dissipate excess energy; however, the generated triplet chlorophyll can transfer its energy to oxygen so that singlet oxygen is produced. Singlet oxygen is a harmful type of reactive oxygen species (ROS) that can cause degradation of membrane and protein structure of photosystems. Plants have several photo-protective mechanisms to protect themselves from excess light. These include chloroplast avoidance movement, reduction of antenna sizes of photosystems, minimizing absorbed energy by non-photochemical quenching (NPQ), reduction of excitation pressure by alternative electron transport directly from PSI to other electron acceptors like oxygen (water-water cycle), photorespiration and PSI cyclic electron flow (under low CO2 conditions), modulation of tetrapyrrole biosynthetic pathways, biosynthesis of antioxidants and efficient repair of damaged photosystems. Any defect in molecular components essential for the light reaction, photo-protection, CO2 assimilation/CCM will be detrimental to photo-autotrophic growth.
Current Research Status
My lab has generated a random nuclear DNA insertional mutant library of C. reinhardtii and has screened it to isolate 21 “interesting” photosynthetic mutants. Out of these 21 mutants, 14 are incapable of photo-autotrophic growth under different light irradiance conditions and seven are pigment deficient but capable of photo-autotrophic growth at a slower rate compared to the parental wild type strain under similar growth conditions. Out of the 14 mutants that are incapable of photo-autotrophic growth, 7 are incapable of photo-autotrophic growth under dim light conditions (15-20 mmol photons m-2s-1), 5 are incapable of photo-autotrophic growth under low-medium light conditions (100-250 mmol photons m-2s-1) and 2 are incapable of photo-autotrophic growth under high light conditions (450-500 mmol photons m-2s-1). Out of the seven pigment deficient mutants that show slow photo-autotrophic growth, 6 are pigment deficient in the light to varying degree and 1 is pigment deficient in the dark but green in the light. We are currently focusing on molecular, biochemical and physiological characterization of these mutants.
Characterization of Chlamydomonas random insertional mutants that are pigment deficient or light sensitive, can lead to the identification of genes that are defective in: (a) sensing light irradiance (b) tolerance to high light stress and (c) making necessary adjustments in their photosynthetic apparatus. Apart from significance of this research in the basic science field, it also has applications in improving solar conversion efficiency in a mass culture. Large chlorophyll antenna leads to over absorption of light by the first few layers of cells in mass culture at a rate that far exceeds the rate at which photosynthesis can utilize them, resulting in dissipation and loss of the excess photons as fluorescence or heat. Meanwhile cells deeper in the culture are deprived of much needed sunlight. Algal strains having a smaller chlorophyll antenna will diminish the over-absorption and wasteful dissipation of excitation energy by the cells and it will also diminish photo-inhibition of photosynthesis at the surface while allowing for greater transmittance of light deeper into the culture. Such altered optical properties of the cells would result in greater photosynthetic productivity and better solar conversion efficiency in the mass culture. Additionally, in Chlamydomonas, NPQ and LhcSR3 is activated in high light, leading to strong heat dissipation even at moderate light. This indicates that improvement of light energy conversion can also be obtained by modulation of heat dissipation response. Our research has the potential of identifying key components in the signal transduction pathway involved in the regulation of Chl biosynthetic pathway and NPQ. This can provide us with tools to improve photosynthetic productivity in future.
B. MICROBIOLOGY PROJECT BASED ON THREE NOVEL BACTERIAL STRAINS WHOSE GENOMES HAVE NOT BEEN SEQUENCED YET.
Our goal is to isolate bacterial strains from natural environment that have the potential to be utilized for biotechnology and bioremediation.
Bacteria can thrive in diverse ecological niches. Diversities in metabolic pathways allow them to survive in these ecological niches. Microbial metabolic diversity has not been fully explored. The metabolic diversity in bacteria can be exploited for removal of harmful chemicals from polluted environment and can also be used for commercial production of important chemicals.
Current Research Status
Three different bacterial strains were isolated from the TAP (Tris Acetate Phosphate) media plates of the green micro-alga Chlamydomonas reinhardtii in our research lab. These bacterial strains were named based on the Chlamydomonas strain they contaminated: LMJSG0182, Clip 185, and CC4533. Several microbiological tests were conducted to characterize the bacteria biochemically. These tests include Gram staining, oxidase test, growth/hemolysis on blood agar media, sugar fermentation tests using Phenol red agar, Mannitol salt agar and MacConkey agar media. LGMSJ0182 grows slowly on LB (Lysogeny Broth) agar media than on TAP agar media. We also tested different carbon sources to find alternative carbon source (other than acetate), for LJMSG0182 in the TAP media. We tested four different antibiotics to identify the best antibiotic and its dose that will be bactericidal but will not inhibit Chlamydomonas growth on TAP media. 16S rRNA gene sequences are used bacterial phylogenetic markers in microbial taxonomy. Partial sequencing of the 16S rRNA gene from these bacterial strains have shown that all three strains are novel strains. Interestingly, LMJSG0182 strain has a novel uncultured bacterial strain as its nearest relative and Acidovorax temperans strain as its second closest relative. Clip185 and cc4533 have 99 % sequence identity to a Microbacterium and Sphingobium strain, respectively. Currently, the nearest relatives do not have sequenced genomes. Additionally, the nearest relatives of these bacterial strains are known to play a role in degradation of polycyclic aromatic hydrocarbons [PAH] (common in insecticides) and polyhydroxyalkanoids [PHA] (commmon in plastics) and removal of heavy metals from polluted water. We plan to perform biochemical tests to check if these bacteria can degrade PAH and PHAs and sequence their whole genomes to identify the metabolic genes that enable these bacteria to degrade PAH and PHA.
Our research findings can open up new avenues for bioremediation of insecticides and plastics from polluted soil and water.
- Invention title:- Suppression of TLA1 gene expression for improved solar conversion efficiency and photosynthetic productivity in plants and algae; found in U.S patent application serial no. 11/423,620 (invention case number 2006-132) filed by UC Berkeley, on June 12, 2006 and issued on June, 29th, 2010. Inventors:- Mautusi Mitra and Anastasios Melis (UC Berkeley)
PATENT ROYALTIES EARNED FROM INVENTION CASE NUMBER 2006-132 TO DATE
- Royalty fee income ($7,875) received from 07-01-2006 till 06-30-2007 from the UC, Berkeley Invention case number 2006-132.
- Royalty fee income ($12,101.22) received from 07-01-2008 till 06-30-2009 from the UC, Berkeley Invention case number 2006-132.
- Royalty fee income ($148.79) received from 07-01-2010 till 06-30-2011 from the UC, Berkeley Invention case number 2006-132.
- Royalty fee income ($443.73.79) received from 07-01-2012 till 06-30-2013 from the UC, Berkeley Invention case number 2006-132.
- Dr. Bernhard Grimm (Former Collaborator, Humboldt University, Berlin, Germany) (past collaborator)
- Dr. Krishna Niyogi (Future Collaborator, UC, Berkeley, California, USA)
- Dr. James V. Moroney (Future Collaborator, Louisiana State University, Louisiana, USA)
- Dr. Terry Bricker (Current Collaborator, Louisiana State University, USA)
- Dr. Anastasios Melis (Future Collaborator, UC, Berkeley, California, USA)
- Dr. Sabeeha Merchant (Future Collaborator, UCLA, California, USA)
Dr. Mitra teaches the following courses at the University of West Georgia:
- BIOL 3134 (Cell and Molecular Biology)
- BIOL1110 (Biodiversity)
- BIOL2107 (Principles of Biology I [for Biology major])
- BIOL6984 (Graduate Research Seminar)
- BIOL4503/6503 (Biochemistry- A Biological perspective)
- BIOL4984 (Senior Seminar for Biology majors)