Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp. strain 6803

Abstract

The existence of active transport systems (permeases) operating on amino acids in the photoautotrophic cyanobacterium Synechocystis sp. strain 6803 was demonstrated by following the initial rates of uptake with 14C-labeled amino acids, measuring the intracellular pools of amino acids, and isolating mutants resistant to toxic amino acids. One class of mutants (Pfa1) corresponds to a regulatory defect in the biosynthesis of the aromatic amino acids, but two other classes (Can1 and Aza1) are defective in amino acid transport. The Can1 mutants are defective in the active transport of three basic amino acids (arginine, histidine, and lysine) and in one of two transport systems operating on glutamine. The Aza1 mutants are not affected in the transport of the basic amino acids but have lost the capacity to transport all other amino acids except glutamate. The latter amino acid is probably transported by a third permease which could be identical to the Can1-independent transport operating on glutamine. Thus, genetic evidence suggests that strain 6803 has only a small number of amino acid transport systems with fairly broad specificity and that, with the exception of glutamine, each amino acid is accumulated by only one major transport system. Compared with heterotrophic bacteria such as Escherichia coli, these permeases are rather inefficient in terms of affinity (apparent Km ranging from 6 to 60 microM) and of Vmax.

Identification of Genes Encoding Amino Acid Permeases by Inactivation of Selected ORFs from the Synechocystis Genomic Sequence

“…Glutamine uptake in strains CSMJ7 and CSMJ8 was most severely affected at 100 µM (∼5% of the wild-type levels) than at 1 µM (∼40%). These results are consistent with previously reported data (Labarre et al 1987;Flores and Muro-Pastor 1990) and suggest that Bgt, which is a high-affinity system for basic amino acids, represents a low-affinity, high-capacity system for glutamine. Uptake of histidine in the bgt mutants was also more severely affected at 100 µM (∼4% of the wild-type levels) than at 1 µM (∼25%).…”

“…Nat is a transport system for neutral amino acids and histidine that seems to be widespread in cyanobacteria (Montesinos et al 1997). In contrast with earlier suggestions (Labarre et al 1987), our results show that Nat also transports glutamine and that it does so with high affinity (K s , 2 µM). Thus, the high-affinity glutamine transport system described by Labarre et al (1987) is Nat itself.…”

“…Glutamine uptake was also severely affected. These results identify the product of sll1270 as an element of the previously described Synechocystis transport system for basic amino acids and glutamine (Labarre et al 1987;Flores and Muro-Pastor 1990). The third element, an ATP-binding subunit, of the E. coli Gln system is GlnQ, and we inactivated the Synechocystis ORF whose putative product is most similar to GlnQ, slr1735.…”

“…strain PCC 6803 shows the highest activity of basic amino acid transport detected for any cyanobacterium and is unique in that it exhibits a high activity of glutamate transport whereas it shows a negligible aspartate transport activity (Labarre et al 1987;Montesinos et al 1997). This cyanobacterium has been described to bear three amino acid transport systems: one for basic amino acids with high affinity for arginine (K s <1 µM; Flores and Muro-Pastor 1990) that would also transport glutamine with low affinity (Labarre et al 1987;Flores and Muro-Pastor 1990); one for neutral (both polar and hydrophobic) amino acids excluding glutamine (Labarre et al 1987); and one for glutamate that would also be a highaffinity glutamine transporter (Labarre et al 1987). The latter system was, however, not supported by further data that indicated the presence of a transport system specific for glutamate and different from the high-affinity glutamine transport system (Montesinos et al 1997).…”

Sodium-Dependent Uptake of Glutamate by Novel ApGltS Enhanced Growth under Salt Stress of Halotolerant CyanobacteriumAphanothece halophytica

“…PCC 6803 is unique because it exhibits high glutamate uptake activity. 20,21) Quintero et al (2001) 9) found that SygltS (slr1145) and Sygtr (sll1102-4) are involved in glutamate uptake in Synechocystis sp. PCC 6803, but the double mutants of these genes reduced uptake activity only to 40-55% of the wild-type values, suggesting the presence of an additional glutamate uptake system.…”