Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis.

The glyoxylate cycle is an anaplerotic pathway of the tricarboxylic acid (TCA) cycle that allows growth on C(2) compounds by bypassing the CO(2)-generating steps of the TCA cycle. The unique enzymes of this route are isocitrate lyase (ICL) and malate synthase (MS). ICL cleaves isocitrate to glyoxylate and succinate, and MS converts glyoxylate and acetyl-CoA to malate. The end products of the bypass can be used for gluconeogenesis and other biosynthetic processes. The glyoxylate cycle occurs in Eukarya, Bacteria and Archaea. Recent studies of ICL- and MS-deficient strains as well as proteomic and transcriptional analyses show that these enzymes are often important in human, animal and plant pathogenesis. These studies have extended our understanding of the metabolic pathways essential for the survival of pathogens inside the host and provide a more complete picture of the physiology of pathogenic micro-organisms. Hopefully, the recent knowledge generated about the role of the glyoxylate cycle in virulence can be used for the development of new vaccines, or specific inhibitors to combat bacterial and fungal diseases.

Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm1021.

The gene encoding pyruvate carboxylase (pyc) was isolated from a Sinorhizobium meliloti Rm1021 cosmid bank by complementation of a Rhizobium tropici pyc mutant. PYC-negative mutants of S. meliloti Rm1021 were isolated by transposon mutagenesis and were unable to grow with glucose or pyruvate as sole carbon sources, but were symbiotically competent in combination with alfalfa plants. PYC activity assays, pyc::lacZ gene fusion studies and an in vivo biotinylation assay showed that PYC activity in S. meliloti was dependent mainly on biotin availability and not on changes in gene transcription. The subunit and holo-enzyme molecular masses of the S. meliloti PYC indicated that the enzyme was an alpha4 homotetramer. The S. meliloti PYC had a high apparent Ka (0.23 mM) for the allosteric activator acetyl-CoA and was product-inhibited by sub-millimolar concentrations of oxaloacetate. In contrast to other bacterial alpha4-PYCs which have been characterized, the S. meliloti enzyme was not strongly inhibited by L-aspartate.

Tricarboxylic acid cycle and anaplerotic enzymes in rhizobia.

Rhizobia are a diverse group of Gram-negative bacteria comprised of the genera Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium and Azorhizobium. A unifying characteristic of the rhizobia is their capacity to reduce (fix) atmospheric nitrogen in symbiotic association with a compatible plant host. Symbiotic nitrogen fixation requires a substantial input of energy from the rhizobial symbiont. This review focuses on recent studies of rhizobial carbon metabolism which have demonstrated the importance of a functional tricarboxylic acid (TCA) cycle in allowing rhizobia to efficiently colonize the plant host and/or develop an effective nitrogen fixing symbiosis. Several anaplerotic pathways have also been shown to maintain TCA cycle activity under specific conditions. Biochemical and physiological characterization of carbon metabolic mutants, along with the analysis of cloned genes and their corresponding gene products, have greatly advanced our understanding of the function of enzymes such as citrate synthase, oxoglutarate dehydrogenase, pyruvate carboxylase and malic enzymes. However, much remains to be learned about the control and function of these and other key metabolic enzymes in rhizobia.

Regulation of pyruvate carboxylase in Rhizobium etli.

Pyruvate carboxylase (PYC) is a biotin-dependent enzyme catalyzing the anaplerotic conversion of pyruvate to oxaloacetate in Rhizobium etli strain CE3. A pyc::Tn5 mutant had severely reduced growth, or failed to grow on sugars, three-carbon organic acids or glycerol, consistent with these substrates being metabolized via pyruvate. Transconjugants expressing a pyc::beta-glucuronidase gene fusion had slightly increased apparent pyc transcription during growth on pyruvate as compared to succinate, similar to the modest carbon source dependent changes in PYC activity reported previously. Biotin supplementation of cultures growing on pyruvate dramatically increased PYC activity but not apparent pyc transcription. Bacteroids isolated from bean nodules did not contain detectable PYC activity while apparent pyc transcription occurred at a moderate level.