Biochemical Validation of the Glyoxylate Cycle in the Cyanobacterium Chlorogloeopsis fritschii Strain PCC 9212

Abstract

Cyanobacteria are important photoautotrophic bacteria with extensive but variable metabolic capacities. The existence of the glyoxylate cycle, a variant of the TCA cycle, is still poorly documented in cyanobacteria. Previous studies reported the activities of isocitrate lyase and malate synthase, the key enzymes of the glyoxylate cycle in some cyanobacteria, but other studies concluded that these enzymes are missing. In this study the genes encoding isocitrate lyase and malate synthase from Chlorogloeopsis fritschii PCC 9212 were identified, and the recombinant enzymes were biochemically characterized. Consistent with the presence of the enzymes of the glyoxylate cycle, C. fritschii could assimilate acetate under both light and dark growth conditions. Transcript abundances for isocitrate lyase and malate synthase increased, and C. fritschii grew faster, when the growth medium was supplemented with acetate. Adding acetate to the growth medium also increased the yield of poly-3-hydroxybutyrate. When the genes encoding isocitrate lyase and malate synthase were expressed in Synechococcus sp. PCC 7002, the acetate assimilation capacity of the resulting strain was greater than that of wild type. Database searches showed that the genes for the glyoxylate cycle exist in only a few other cyanobacteria, all of which are able to fix nitrogen. This study demonstrates that the glyoxylate cycle exists in a few cyanobacteria, and that this pathway plays an important role in the assimilation of acetate for growth in one of those organisms. The glyoxylate cycle might play a role in coordinating carbon and nitrogen metabolism under conditions of nitrogen fixation.

Keywords: TCA cycle; acetyl coenzyme A (acetyl-CoA); cyanobacteria; glyoxylate cycle; isocitrate lyase; malate synthase; metabolism; photosynthesis; poly-3-hydroxybutyrate; tricarboxylic acid cycle (TCA cycle) (Krebs cycle).

FIGURE 1.

Scheme showing the glyoxylate and TCA cycles in some cyanobacteria. Abbreviations used were: 2-OG, 2-oxoglutarate; 2-OGDC, 2-oxoglutarate decarboxylase; ACO, aconitase; CS, citrate synthase; FUM, fumarase; ICL, isocitrate lyase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; MS, malate synthase; PDH, pyruvate dehydrogenase; SDH, succinic acid dehydrogenase; SSA, succinic semialdehyde; SSADH, succinic semialdehyde dehydrogenase. The heavy arrows show the two reactions specific for the glyoxylate cycle.

FIGURE 2.

Characterization of purified recombinant fumarase (SYNPCC7002_A2041). A, SDS-PAGE (lane 1) and immunoblotting analysis with a commercial antibody to the poly-His₆ tag (lane 2) of the purified fumarase. B, HPLC analysis showing that malate (peak 1) was converted to fumarate (peak 2) by fumarase (SYNPCC7002_A2041). Specifically, when the product of SynPCC7002_A2041 was incubated with 10 mm malate at room temperature, 2.1 mm fumarate was formed and 2.2 mm malate was consumed. C, HPLC analysis showing the formation of malate (peak 1) from fumarate (peak 2) catalyzed by the purified fumarase (SYNPCC7002_A2041). When the product of SynPCC7002_A2041 was incubated with 2.5 mm fumarate at room temperature, 1.9 mm malate was formed and 2.1 mm fumarate was consumed (C). The differences in the peak areas for identical amounts of fumarate and malate are due to the very different molar extinction coefficients of these two compounds at 210 nm. Insets represent the enlarged parts of the elution curves from 11 to 16 min to illustrate the changes observed more clearly. Other details of the assay conditions are described under ”Experimental Procedures.“

FIGURE 3.

Characterizations of purified recombinant isocitrate lyase (UYEDRAFT_02681) and malate synthase (UYEDRAFT_02682). A, SDS-PAGE and immunoblotting analysis with a commercial antibody to the poly-His₆ tag for isocitrate lyase (ICL) and malate synthase (MS). B, HPLC analysis showing that isocitrate (peak 1) was converted to glyoxylate (peak 2) and succinate (peak 3) by the purified isocitrate lyase (UYEDRAFT_02681). When the protein product of UYEDRAFT_02681 was incubated with 2 mm isocitrate, 0.3 mm isocitrate was consumed and 0.25 mm succinate and 0.27 mm glyoxylate were produced. C, HPLC analysis showing the formation of malate (peak 5) from glyoxylate (peak 2) and acetyl-CoA (peak 4) catalyzed by purified malate synthase (UYEDRAFT_02682). Specifically, when the protein product from ORF UYEDRAFT_02682 was incubated with 2 mm glyoxylate and 2 mm acetyl-CoA, 1.2 mm glyoxylate and 1.1 mm acetyl-CoA were consumed, and 0.9 mm malate was produced. D, HPLC analysis showing production of isocitrate (peak 1) from glyoxylate (peak 2) and succinate (peak 3) catalyzed by the purified isocitrate lyase. Specifically, 0.15 mm isocitrate was produced, and 0.19 mm glyoxlyate and 0.15 mm succinate were consumed, when 1 mm succinate and 1 mm glyoxylate were incubated with the product of UYEDRAFT_02681. The large differences in the peak area for identical amounts of acetyl-CoA and glyoxylate are due to the different molar extinction coefficients of these two compounds at 210 nm. Detailed assay conditions are described under ”Experimental Procedures.“

FIGURE 4.

Acetate assimilation and growth curves for C. fritschii PCC 9212. Black lines indicate the cell density and gray lines indicate the acetate concentrations in the medium at different times during the batch growth cycle. A, C. fritschii PCC 9212 growing under standard conditions. B, C. fritschii PCC 9212 growing under low CO₂ conditions (cultures were sparged with air). C, C. fritschii PCC 9212 grown under dark conditions (1% CO₂ in air). The data shown are averages of three biological replicates, and the error bars show the standard deviation. Other details concerning the growth conditions are described under ”Experimental Procedures.“

FIGURE 5.

Verification of the presence of glyoxylate cycle genes (aceBA) in Synechococcus sp. PCC 7002 strain 7002-glyox by PCR. The template DNA was derived from wild-type C. fritschii PCC 9212 (lane 9212), from wild-type Synechococcus sp. PCC 7002 (lane 7002), and from the recombinant strain 7002-glyox (lane 7002-glyox), which has the aceBA genes from C. fritschii PCC 9212 inserted in plasmid pAQ1-Ex as described under ”Experimental Procedures.“

FIGURE 6.

Acetate assimilation and growth analysis of Synechococcus sp. PCC 7002. Black lines indicate cell density and gray lines indicate the acetate concentrations in the medium at different growth stages. A, Synechococcus sp. PCC 7002 grown under standard conditions; B, Synechococcus sp. PCC 7002 growing under low light conditions; C, Synechococcus sp. PCC 7002 growing under dark conditions; D, Synechococcus sp. PCC 7002 growing under low CO₂ conditions. WT, wild type Synechococcus sp. PCC 7002; 7002-glyox, Synechococcus sp. PCC 7002 strain with aceBA genes of C. fritschii PCC 9212 expressed from pAQ1. The data shown are averages of three biological replicates, and the error bars show the S.D.

FIGURE 7.

Relative transcript abundances for mRNAs in C. fritschii PCC 9212 grown with or without acetate. A, gene neighborhood around the glyoxylate cycle genes. Numbers above each gene indicate the fold-difference of mRNA abundance in cells grown with acetate compared with cells grown without acetate. B, scatter plot showing the relative abundance of all the mRNAs under growth conditions without acetate (9212) or with acetate (9212A). Gray lines indicate a 2-fold increase or 50% decrease in mRNA level. aceA, isocitrate lyase; aceB, malate synthase; phaA, acetyl-CoA acetyltransferase; phaB, acetoacetyl-CoA reductase; phaE, poly(R)-hydroxyalkanoic acid synthase, class III, PhaE subunit; phaC, poly(R)-hydroxyalkanoic acid synthase, class III, PhaC subunit; phaZ, poly(3-hydroxybutyrate) depolymerase; ppsA, phosphoenolpyruvate synthase.

FIGURE 8.

Accumulation of PHB in C. fritschii PCC 9212. PHB contents of C. fritschii PCC 9212 were monitored as a function of batch growth under standard conditions for C. fritschii PCC 9212. The bars indicate the total cell dry weight (CDW), and the black portions of the bars show the PHB content at different growth stages. This information is also plotted to emphasize the kinetics of PHB production. In the absence of added acetate, PHB accounted for ∼5% of total CDW. The inset shows that CDW changed only slightly when 10 mm acetate was added to the medium, but PHB accumulated to a much higher level, ∼15% of total CDW. The data shown are averages of three biological replicates, and the error bars show the S.D.