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Title: Nitrogen-Dependent Carbon Fixation by Picoplankton In Culture and in the Mississippi River

Abstract

The pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC), of the marine cyanobacterium Synechococcus PCC 7002, was isolated and sequenced. PEPC is an anaplerotic enzyme, but it may also contribute to overall CO2 fixation through β-carboxylation reactions. A consensus sequence generated by aligning the pepc genes of Anabaena variabilis, Anacystis nidulans and Synechocystis PCC 6803 was used to design two sets of primers that were used to amplify segments of Synechococcus PCC 7002 pepc. In order to isolate the gene, the sequence of the PCR product was used to search for the pepc nucleotide sequence from the publicly available genome of Synechococcus PCC 7002. At the time, the genome for this organism had not been completed although sequences of a significant number of its fragments are available in public databases. Thus, the major challenge was to find the pepc gene among those fragments and to complete gaps as necessary. Even though the search did not yield the complete gene, PCR primers were designed to amplify a DNA fragment using a high fidelity thermostable DNA polymerase. An open reading frame (ORF) consisting of 2988 base pairs coding for 995 amino acids was found in the 3066 bp PCR product. The pepc genemore » had a GC content of 52% and the deduced protein had a calculated molecular mass of 114,049 Da. The amino acid sequence was closely related to that of PEPC from other cyanobacteria, exhibiting 59-61% identity. The sequence differed significantly from plant and E. coli PEPC with only 30% homology. However, comparing the Synechococcus PCC 7002 sequence to the recently resolved E. coli PEPC revealed that most of the essential domains and amino acids involved in PEPC activity were shared by both proteins. The recombinant Synechococcus PCC 7002 PEPC was expressed in E. coli.« less

Authors:
; ;
Publication Date:
Research Org.:
Howard University, Washington, DC
Sponsoring Org.:
USDOE
OSTI Identifier:
900483
Report Number(s):
DOE/ER/62453- Final Report
DOE Contract Number:  
FG02-97ER62453
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Synechococcus PCC 7002, physophoenolpyruvate carboxylase, pepc gene, cyanobacteria

Citation Formats

Smith, Aubrey, Coomes, Marguerite W, and Smith, Thomas E. Nitrogen-Dependent Carbon Fixation by Picoplankton In Culture and in the Mississippi River. United States: N. p., 2005. Web. doi:10.2172/900483.
Smith, Aubrey, Coomes, Marguerite W, & Smith, Thomas E. Nitrogen-Dependent Carbon Fixation by Picoplankton In Culture and in the Mississippi River. United States. https://doi.org/10.2172/900483
Smith, Aubrey, Coomes, Marguerite W, and Smith, Thomas E. Sat . "Nitrogen-Dependent Carbon Fixation by Picoplankton In Culture and in the Mississippi River". United States. https://doi.org/10.2172/900483. https://www.osti.gov/servlets/purl/900483.
@article{osti_900483,
title = {Nitrogen-Dependent Carbon Fixation by Picoplankton In Culture and in the Mississippi River},
author = {Smith, Aubrey and Coomes, Marguerite W and Smith, Thomas E},
abstractNote = {The pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC), of the marine cyanobacterium Synechococcus PCC 7002, was isolated and sequenced. PEPC is an anaplerotic enzyme, but it may also contribute to overall CO2 fixation through β-carboxylation reactions. A consensus sequence generated by aligning the pepc genes of Anabaena variabilis, Anacystis nidulans and Synechocystis PCC 6803 was used to design two sets of primers that were used to amplify segments of Synechococcus PCC 7002 pepc. In order to isolate the gene, the sequence of the PCR product was used to search for the pepc nucleotide sequence from the publicly available genome of Synechococcus PCC 7002. At the time, the genome for this organism had not been completed although sequences of a significant number of its fragments are available in public databases. Thus, the major challenge was to find the pepc gene among those fragments and to complete gaps as necessary. Even though the search did not yield the complete gene, PCR primers were designed to amplify a DNA fragment using a high fidelity thermostable DNA polymerase. An open reading frame (ORF) consisting of 2988 base pairs coding for 995 amino acids was found in the 3066 bp PCR product. The pepc gene had a GC content of 52% and the deduced protein had a calculated molecular mass of 114,049 Da. The amino acid sequence was closely related to that of PEPC from other cyanobacteria, exhibiting 59-61% identity. The sequence differed significantly from plant and E. coli PEPC with only 30% homology. However, comparing the Synechococcus PCC 7002 sequence to the recently resolved E. coli PEPC revealed that most of the essential domains and amino acids involved in PEPC activity were shared by both proteins. The recombinant Synechococcus PCC 7002 PEPC was expressed in E. coli.},
doi = {10.2172/900483},
url = {https://www.osti.gov/biblio/900483}, journal = {},
number = ,
volume = ,
place = {United States},
year = {2005},
month = {4}
}