Phenotypic Complementation of High CO₂-Requiring Mutants of the Cyanobacterium Synechococcus sp. Strain PCC 7942 by Inosine 5′-Monophosphate

Abstract

The high C02-requiring mutants of Synechococcus PCC 7942, D4 and R14, were obtained by deletion or inactivation (respectively) of an open reading frame immediately downstream of rbc (the operon encoding the subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase). These mutants exhibit photosynthetic characteristics similar to those of high C02-grown wild type, unlike other cyanobacterial high C02-requiring mutants, where the apparent photosynthetic affinity for inorganic carbon is approximately 2 orders of magnitude lower than that of the wild type. Sequence analysis and metabolic complementation of the mutants by inosine 5'-monophosphate identified this open reading frame as the cyanobacterial equivalent of purK, the eubacterial gene encoding subunit 11 of phosphoribosyl aminoimidazole carboxylase in the purine biosynthetic pathway. Exposure of high C02-grown Synechococcus to low CO2 conditions led to the induction of transcription of purK. It is suggested that the high C02-requiring phenotype of these mutants resulted from the defect in purine biosynthesis after exposure to low CO2. We also raise the possibility that the level of cellular purines is involved in the process of adaptation of cyanobacteria to low concentrations of CO2.as tools for the elucidation of the physiological and molecular basis of the Ci-concentrating mechanism. Most of these mutants exhibit an apparent photosynthetic affinity approximately 2 orders of magnitude lower than that of high C02-grown wild type (5, 7, 9, 10, 12, 15-17, 19, 22, 25). High C02-requiring mutants in which the photosynthetic characteristics are similar to those observed in wild-type cells grown under high C02 were isolated from Chlamydomonas reinhardtii (13) and Synechococcus sp. PCC 7942 (10). The mutant JR12 of the latter was obtained after transformation of the wild type with a construct designed to modify the genomic region of rbc. This mutant resulted from a rare recombination event in which the integration of the modified DNA into the genome occurred by single crossover, but part of the plasmid (1.7 kb) and about 2 kb downstream of rbc were deleted (10). The performance of this mutant indicated the significance of the genomic region downstream of rbc with regard to the ability of cyanobacteria to grow in the presence of low ambient C02 concentration. In this report, we present physiological and molecular characteristics of mutants obtained following specific modifications of this region. MATERIALS AND METHODSCyanobacteria can grow under a wide range of external Ci2 concentrations even though the Km(C02) of their Rubisco is some 20-fold higher than the concentration of dissolved C02 present in media at equilibrium with air (2). When transferred from high-to low-CO2 conditions, the cells undergo an adaptation process that includes the induction of a Ci-concentrating mechanism. As a consequence of this increase in the ability to accumulate Ci internally, the apparent photosynthetic affinity for extemal Ci is 10-to 20-fold higher in low than in high C...

Biogenesis and Ultrastructure of Carboxysomes from Wild Type and Mutants of Synechococcus sp. Strain PCC 7942

“…The preponderance of the immature forms means that development of carboxysomes is slowed down in D4, which explains its HCRM phenotype. An alternative explanation is the predicted decrease in cellular purine levels under low CO, (Schwarz et al, 1992a). We cannot provide any evidence to link the failure in purine biosynthesis with that of carboxysome assembly.…”

“…Vol. 107, 1995 Kanamycin-resistant revertant of HCRM RK12, which had the same deletion and phenotype as D4 Marco et al, 1993Schwarz et al, 1992bLieman-Hurwitz et al, 1991Schwarz et al, 1992aLieman-Hurwitz et al, 1991Lieman-Hurwitz et al, 1991 under 110 piE m~2 s" 1 continuous illumination, at 28°C, and bubbled with 5% (v/v) CO 2 -enriched air (high CO 2 ). When indicated, culture aeration was changed to air bubbling (low CO 2 ) for 6, 12, or 24 h before collection.…”

“…The ORF involved is located immediately downstream of rbcLS (Liemann-Hurwitz et al, 1991) and shows significant homology with bacterial purK (Schwarz et al, 1992a), a subunit of phosphoribosyl aminoamidazole carboxylase that is required for the operation of the enzyme only under low-CO, conditions. Neither the capacity to accumulate Ci nor the apparent photosynthetic affinity for external Ci are seriously affected in this mutant (Schwarz et al, 1992a). We have observed that most of its carboxysomes are immature electron-translucent ones, but that it also exhibits carboxysomes with an electron-translucent area and the typical polyhedral bodies.…”

The photorespiratory glycolate metabolism is essential for cyanobacteria and might have been conveyed endosymbiontically to plants

“…In this respect, the double mutant ⌬glcD1/⌬glcD2 resembled the ⌬purK mutant of Synechococcus sp. strain PCC 7942, where the HCR phenotype emerged from an inability to produce purines under LC (31). Last, after exposure of mutant ⌬glcD1/⌬glcD2 to LC for 6 h, the photosynthetic K 1/2 (C i ) decreased considerably as in the WT (Fig.…”

Isolation of cyanobacterial mutants exhibiting growth defects under microoxic conditions by transposon tagging mutagenesis of <i>Synechocystis</i> sp. PCC 6803

“…A purK-lacking mutant was isolated from another cyanobacterium Synechococcus elongatus PCC 7942 (Schwarz et al, 1992). The purK mutant, R14, showed a high-CO 2 -requiring phenotype.…”