Functional genomics of the regulation of the nitrate assimilation pathway in Chlamydomonas

Abstract

The existence of mutants at specific steps in a pathway is a valuable tool of functional genomics in an organism. Heterologous integration occurring during transformation with a selectable marker in Chlamydomonas (Chlamydomonas reinhardtii) has been used to generate an ordered mutant library. A strain, having a chimeric construct (pNia1::arylsulfatase gene) as a sensor of the Nia1 gene promoter activity, was transformed with a plasmid bearing the paramomycin resistance AphVIII gene to generate insertional mutants defective at regulatory steps of the nitrate assimilation pathway. Twenty-two thousand transformants were obtained and maintained in pools of 96 for further use. The mutant library was screened for the following phenotypes: insensitivity to the negative signal of ammonium, insensitivity to the positive signal of nitrate, overexpression in nitrate, and inability to use nitrate. Analyses of mutants showed that (1) the number or integrated copies of the gene marker is close to 1; (2) the probability of cloning the DNA region at the marker insertion site is high (76%); (3) insertions occur randomly; and (4) integrations at different positions and orientations of the same genomic region appeared in at least three cases. Some of the mutants analyzed were found to be affected at putative new genes related to regulatory functions, such as guanylate cyclase, protein kinase, peptidyl-prolyl isomerase, or DNA binding. The Chlamydomonas mutant library constructed would also be valuable to identify any other gene with a screenable phenotype.

Figure 1.

Scheme of steps followed for the construction of the Chlamydomonas mutant library.

Figure 2.

Screening procedure of mutants. From a 96-well microtiter plate, 3 replicates were done on different square plates containing the indicated nitrogen sources. ARS activity test was performed over plates A and C. NI and ON mutants were screened from plates A (yellow and green rings, respectively), whereas AI mutants were screened from plate C (red rings). Nitrate assimilation mutants (Nit⁻) were observed over plate B (white rings). Examples of each phenotype are shown magnified.

Figure 3.

ARS activity in the AI mutants. Cells were grown in TAP-ammonium-nitrate media and measurements of the ARS activity were performed after 3 to 4 d of growth. ARS activity is determined as absorbance units per cell number of each culture and expressed as relative folds with respect to activity in the parental strain 704 under the same conditions. Data correspond to four independent determinations for each mutant.

Figure 4.

Determination by Southern blot of marker gene copy number. Genomic DNAs from 18 randomly isolated paramomycin-resistant mutants were digested with PstI. Asterisks indicate the specific hybridization bands corresponding to AphVIII insertions. Triangles indicate two unspecific hybridization bands present in all the mutants. M, Lane of DNA size marker.

Figure 5.

Schematic representation of the localization of three different linked mutations. Triangles represent the point of insertion and arrows the direction of the plasmid indicating the beginning of the putative deletions for each mutant. Map images of the genomic sequences are from the JGI Chlamydomonas genome server for the corresponding scaffold numbers: 48 (A), 3 (B), and 49 (C).