Vitamin B12-mediated physiology, mutualisms, and gene regulation studies in Chlamydomonas reinhardtii and Phaeodactylum tricornutum
Many organisms engage in symbiotic relationships that govern, to varying degrees, the function of ecosystems and their evolutionary history. It is important to understand the origins and dynamics of these systems. Especially important is the aquatic ecosystem, which is essential for approximately 50% of global primary production, and where many photosynthetic algae participate in symbiosis with bacteria where the latter provide vitamins such as cobalamin (vitamin B12) in exchange for fixed carbon from the algae. Vitamin B12 is used as a cofactor by methionine synthase (METH), but many algae such as the freshwater chlorophyte, Chlamydomonas reinhardtii, and the marine diatom, Phaeodactylum tricornutum, also have a B12-independent form of methionine synthase (METE). This means that they can utilize METH if B12 is available, but when it is absent or in limited supply, they use METE. To study the influence of vitamin exchange, CRISPR (clustered regularly interspaced short palindromic repeats) was used to make gene-edited metE mutants of both species. This enabled the study of the physiology of newly B12 dependent algae and mutualisms with B12-producing bacteria. As well as the role of B12 as a cofactor, it also acts to repress gene expression. Most notably, METE shows rapid and robust repression in the presence of B12 in both C. reinhardtii and P. tricornutum. This has been previously shown to occur through the promoter regions in both organisms. This dissertation has three main parts which are i) physiological and genetic consequences of acquired vitamin auxtrophy in C. reinhardtii and P. tricornutum; ii) bacterial mutualisms underpinned with B12 in C. reinhardtii and P. tricornutum; and iii) a comparative study of the regulation of C. reinhardtii and P. tricornutum METE using fluorescent reporters. To understand the physiological consequences of vitamin auxotrophy, the B12 requirement and response characteristics were determined for growth, growth rate, cell size changes, and transcript response in vitamin deplete and replete conditions. The B12-dependent C. reinhardtii had a higher vitamin requirement than P. tricornutum. When looking at cell size, C. reinhardtii was shown to drastically increase size in vitamin deprivation, which did not occur with P. tricornutum. One-carbon (C1) metabolism, which is important for amino acid (methionine) synthesis via METE or METH, nucleotide synthesis, and broader methylation patterns, was next investigated in C. reinhardtii and P. tricornutum. Observed were higher differential expression of C1 cycle transcripts in both C. reinhardtii and P. tricornutum metE mutants under vitamin deprivation, which may be a result of altered metabolic capabilities. Comparing METE genes between related species in C. reinhardtii and P. tricornutum revealed that Edaphochlamys debaryana and Fistulifera solaris have two METE genes. The B12 dependent C. reinhardtii was able to form a mutualism with B12-producing bacteria Mesorhizobium loti and Rhodospirillum rubrum. An RNA sequencing experiment was conducted with samples taken just after initiation of a co-culture and then after subculturing once. Clean reads ranging from 53-81 million were obtained with ~95% coverage in all samples. Differentially expressed genes (DEGs) were found, including in ‘biosynthesis of secondary metabolites’, ‘oxidation-reduction process’, and ‘transporter activity’. Of note was that distinct differential expression patterns were seen in co-cultures with M. loti versus R. rubrum, and a distinct B12 deprivation response was apparent in the latter. B12-dependent P. tricornutum in co-culture with B12 producing Halomonas sp. did not form a stable mutualism. Comparative analysis of the transcriptional regulation of METE in C. reinhardtii and P. tricornutum was carried out by using respective METE promoters (PMETE) to drive expression of fluorescent reporter mVenus. The findings showed crucial differences between the reporters in terms of B12 dose response, timing, and repression via B12 analogues. When the reporter strains were co-cultured with B12-producing bacteria M. loti repressed expression of mVenus in C. reinhardtii but Halomonas sp. did not in P. tricornutum, indicating that B12 was not supplied to the alga in the latter case. Diurnal datasets were also investigated and found that METE in C. reinhardtii is under strict diurnal control, whereas the P. tricornutum METE exhibits higher expression through the dark cycle. However, when P. tricornutum PMETE-mVenus reporters were assayed over the diurnal cycle, there was no change in fluorescent expression, suggesting the diurnal regulation may not be through the promoter.
Mutualisms, B12 regulation, Chlamydomonas reinhardtii, Phaeodactylum tricornutum
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