Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

Doron Amichay, Ruth Levitz, Michael Gurevitz

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803†rbc, were detected within 8 days when grown under 5% CO2 in air. These transformants were unable to grow in air (0.03% CO2). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO2 in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803Δrbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.

Original languageEnglish (US)
Pages (from-to)465-476
Number of pages12
JournalPlant Molecular Biology
Volume23
Issue number3
DOIs
StatePublished - Nov 1993

Fingerprint

Ribulose-Bisphosphate Carboxylase
Oxygenases
ribulose-bisphosphate carboxylase
oxygenases
mutants
Enzymes
enzymes
Genes
Bearings (structural)
operon
Operon
Air
air
carbon dioxide
Chloramphenicol
chloramphenicol
genes
loci
Rhodospirillum rubrum
Chloramphenicol Resistance

Keywords

  • carboxysomes
  • cyanobacteria
  • rbc genes
  • Rubisco
  • transformation

ASJC Scopus subject areas

  • Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes. / Amichay, Doron; Levitz, Ruth; Gurevitz, Michael.

In: Plant Molecular Biology, Vol. 23, No. 3, 11.1993, p. 465-476.

Research output: Contribution to journalArticle

@article{f638707de82948d28242b694f306c375,
title = "Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes",
abstract = "The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803†rbc, were detected within 8 days when grown under 5{\%} CO2 in air. These transformants were unable to grow in air (0.03{\%} CO2). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5{\%} CO2 in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803Δrbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.",
keywords = "carboxysomes, cyanobacteria, rbc genes, Rubisco, transformation",
author = "Doron Amichay and Ruth Levitz and Michael Gurevitz",
year = "1993",
month = "11",
doi = "10.1007/BF00019295",
language = "English (US)",
volume = "23",
pages = "465--476",
journal = "Plant Molecular Biology",
issn = "0167-4412",
publisher = "Springer Netherlands",
number = "3",

}

TY - JOUR

T1 - Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

AU - Amichay, Doron

AU - Levitz, Ruth

AU - Gurevitz, Michael

PY - 1993/11

Y1 - 1993/11

N2 - The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803†rbc, were detected within 8 days when grown under 5% CO2 in air. These transformants were unable to grow in air (0.03% CO2). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO2 in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803Δrbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.

AB - The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803†rbc, were detected within 8 days when grown under 5% CO2 in air. These transformants were unable to grow in air (0.03% CO2). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO2 in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803Δrbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.

KW - carboxysomes

KW - cyanobacteria

KW - rbc genes

KW - Rubisco

KW - transformation

UR - http://www.scopus.com/inward/record.url?scp=0027691221&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027691221&partnerID=8YFLogxK

U2 - 10.1007/BF00019295

DO - 10.1007/BF00019295

M3 - Article

VL - 23

SP - 465

EP - 476

JO - Plant Molecular Biology

JF - Plant Molecular Biology

SN - 0167-4412

IS - 3

ER -