The 5'-flanking region of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is crucial for growth of the cyanobacterium Synechococcus sp. strain PCC 7942 at the level of CO2 in air

Abstract

Transformation of the high-CO2-requiring mutants (hcr) 0221 and El derived from the cyanobacterium Synechococcus sp. strain PCC 7942 by a wild-type DNA library restored their ability to grow at the level of CO2 in air. A plasmid (pE12) containing a 10-kilobase DNA insert was rescued from a 0221 heterogenote and proved to transform both 0221 and El to the wild-type phenotype. The capacity of the pE12 subclones to confer the wild-type phenotype to 0221 transformants enabled the mapping of the mutation in 0221 (designated hcrO221) within a 232-base-pair PstI-BstXI DNA restriction fragment. Sequence analysis revealed two open reading frames (ORFs) at positions -1745 to -1262 (ORFI) and -1218 to -393 (ORFiH) upstream of the rbcL gene. A 3-kilobase PstI fragment of 0221 was cloned, and hcrO221 was found to be a point mutation within the PstI-BstXI region -1309 nucleotides upstream of the rbcL gene. The significance of this flanking region for adaptation to air levels of CO2 was further demonstrated by the generation of new hcr mutants following insertional inactivation of wild-type DNA in the BstXI site. Electron microscopy revealed aberrant carboxysome structures in growing cells of the hcr mutants, a defect that was possibly related to the mutation, since transformation with pE12 derivatives restored the carboxysome structure to normal.The photosynthetic performance of photosynthetic organisms is limited by the availability of CO2 in air to ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase because of the low affinity of the carboxylase for CO2 and the competition between CO2 and 02 for the reaction with RuBP (2, 3, 6). Cyanobacteria and green algae possess an inducible mechanism for concentrating inorganic carbon (C,) internally to levels high enough for efficient carboxylation (1,3,4,(11)(12)(13). This mechanism enables them to adapt to the level of CO2 in air (0.03%) and to grow photoautotrophically in this environment (12,15). During the adaptation process, the photosynthetic affinity to external Ci (3,12,16), the rate of C, transport (3, 12, 15, 17), and the synthesis of a 42-kilodalton cytoplasmic membrane protein (19,20) are increased. Furthermore, cyanobacteria growing at the air level of CO2 contain more carboxysomes than those growing at 5% CO2 in air do (30). In both cyanobacteria and photosynthetic bacteria, these bodies contain a significant amount of RuBP carboxylase (5, 6). It has been suggested that this subcellular location of the enzyme is important for the efficient utilization of CO2 (22).In an attempt to elucidate the molecular basis of the adaptation process, we have isolated mutants from Synechococcus sp. strain PCC 7942 that failed to adapt to the air level of CO2 (15, 25) but that grew normally at 5% CO2 in air. In these mutants the utilization of the internal Ci pool for photosynthesis is altered, while the accumulation of C, appears to be normal (15,21,25). Molecular analysis of these mutants is a valuable tool for identifying the putative gene(s) and product(s) involved in the adaptat...

Physiological and Molecular Aspects of the Inorganic Carbon-Concentrating Mechanism in Cyanobacteria

“…Such mutants would be expected to exhibit normal photosynthetic performance under high-CO2 conditions (including apparent photosynthetic affinity for CQ), but would fail to adapt and grow under low CO2. This behavior contrasts with that of other types of high-C02-requiring mutants defective in the ability to utilize the internal C1 pool, which exhibit very low apparent photosynthetic affinity to C1 (8) . The level of some of the transcripts originating from this region (including that from rbc) depends on the CO2 concentration during growth.…”

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

“…It would be expected that immature stages appear during this period of active assembly of new carboxysomes. We have also observed carboxysomes with an electron-translucent area (type C) in Anabaena cells grown under severe limitation of Q induced by Na + deficiency (E. FernandezValiente, personal communication), in Synechococcus (Friedberg et al, 1989;Kaplan, 1990), and in Synechocystis (Ogawa et al, 1994), although such structures had not been pointed out by the various authors cited.…”

“…Such increased synthesis of carboxysome precursors might result in their assembly into the bar-shaped carboxysomes frequently found in this mutant. Bar-shaped carboxysomes have been reported in HCRM type I and PVU (Price and Badger, 1991) and E1 (Friedberg et al, 1989) and have been interpreted as aberrant bodies resulting from the alteration of ccmL. However, this type of carboxysome coexists with typical carboxysomes in the same cell and have also been found in wildtype Synechococcus under conditions of predictable severe stress for C , such as stationary-phase old cultures (Gantt and Conti, 1969) and cultures bubbled with 30 pL of CO, in air at pH 9.5 (McKay et al, 1993).…”

“…Severa1 mutants in which the requirement for high CO, for growth coincides with anomalous carboxysomes have been described. A mutant of Synechococcus PCC 7942 P/N (Price and Badger, 1991) lacks carboxysomes, whereas 0221 (Friedberg et al, 1989) and N1 show diffuse or aberrant carboxysomes. The genomic lesions map within three of the ORFs identified upstream of the region of the rbcLS operon, namely ccmM, ccmN, and ccmO.…”

Mutants of Synechocystis PCC6803 Defective in Inorganic Carbon Transport

“…Mutants of similar phenotype have been isolated from Synechococcus PCC7942 (1, 11, 14, 21, 23). Molecular analysis of two of these mutants of Synechococcus, 022, and E1, revealed that the sites of mutations are on the 5'-flanking region of the gene encoding the large subunit of ribulose-1 ,5-bisphosphate carboxylase/oxygenase (8). Price and Badger (21) (Fig.…”