Summary The rice resistance gene Xa21 confers resistance against the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). The molecular genetic mechanism controlling the integration of the Xa21‐mediated disease resistance response with the developmental program in rice is under study in this model system. Reproducible means of infecting plants at certain developmental stages were designed based on the timing of full expansion of the leaf. Xa21‐resistance progressively increases from the susceptible juvenile leaf 2 stage through later stages, with 100% resistance at the adult leaf 9/10 stage. We found that Xa21 expression is independent of plant developmental stage, infection with Xoo, or wounding. Expression of the Xa21 gene transcript is not correlated with expression of Xa21 disease resistance indicating that the developmental regulation of Xa21‐resistance is either controlled post‐transcriptionally or by other factors.

Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) catalyzes the irreversible β‐carboxylation of phosphoenolpyruvate (PEP) to yield oxaloacetate (OAA) and inorganic phosphate (Pi). PEPC is found in higher plants and most types of bacteria including cyanobacteria. Several PEPCs have been purified from plants and bacteria, including thermophilic bacteria. PEPC contributes to photosynthetic and anaplerotic CO2 fixation. In this study we computed the physicochemical properties of cyanobacterial PEPCs and compared sequences from fresh water and marine sources. The alignment of cyanobactyerial enzymes and secondary structure analysis revealed that there are conserved amino acid substitutions and polymorphisms between PEPCs from marine and fresh water organisms. Furthermore, some marine subgroups seem to possess unique amino acid stretches that may modulate various aspects of catalysis and regulation. Phosphoenolpyruvate carboxylase from Synechococcus PCC 7002, a marine organism, most closely resembles PEPC from fresh water organisms; for this reason, the enzyme was chosen for homology modeling alongside PEPCs from the fresh water strain Anabaena variabilis and the marine cyanobacterium Synechococcus RS 9917. The physicochemical characteristics and the 3D models provide a framework for the purification and characterization of cyanobacterial PEPCs.

ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) is a key catabolic enzyme found in various species of bacteria, plants, and yeast. PEPCK may play a role in carbon fixation in aquatic ecosystems consisting of photosynthetic cyanobacteria. RuBisCO-based CO2fixation is prevalent in cyanobacteria through C3intermediates; however, a significant amount of carbon flows into C4acids during cyanobacterial photosynthesis. This indicates that a C4mechanism for inorganic carbon fixation is prevalent in cyanobacteria with PEPCK as an importantβ-carboxylation enzyme. Newly available genomic information has confirmed the existence of putativePEPCKgenes in a number of cyanobacterial species. This project represents the first structural and physicochemical study of cyanobacterial PEPCKs. Biocomputational analyses of cyanobacterial PEPCKs were performed and a homology model ofCyanothecesp. PCC 7424 PEPCK was generated. The modeled enzyme consists of an N-terminal and C-terminal domains with a mixedα/βtopology with the active site located in a deep cleft between the two domains. Active site residues and those involved in metal ion coordination were found to be conserved in the cyanobacterial enzymes. An active site lid which is known to close upon substrate binding was also predicted. Amino acid stretches that are unique to cyanobacterial PEPCKs were also identified.

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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 CO 2 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.