Interactions of DNA with Biofilm-Derived Membrane Vesicles

The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs) and DNA, both matrix components, demonstrated concentration-, pH-, and cation-dependent interactions. Furthermore, MV-DNA association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction of matrix polymers and other constituents.The biofilm matrix contributes to the chemistry, structure, and function of biofilms and is crucial for the development of fundamental biofilm properties (46, 47). Early studies defined polysaccharides as the matrix component, but proteins, lipids, and nucleic acids are all now acknowledged as important contributors (7, 15). Indeed, DNA has emerged as a vital participant, fulfilling structural and functional roles (1, 5, 6, 19, 31, 34, 36, 41, 43, 44). The phosphodiester bond of DNA renders this polyanionic at a physiological pH, undoubtedly contributing to interactions with cations, humic substances, fine-dispersed minerals, and matrix entities (25, 41, 49).In addition to particulates such as flagella and pili, membrane vesicles (MVs) are also found within the matrices of gram-negative and mixed biofilms (3, 16, 40). MVs are multifunctional bilayered structures that bleb from the outer membranes of gram-negative bacteria (reviewed in references 4, 24, 27, 28, and 30) and are chemically heterogeneous, combining the known chemistries of the biofilm matrix. Examination of biofilm samples by transmission electron microscopy (TEM) has suggested that matrix material interacts with MVs (Fig. ​(Fig.1).1). Since MVs produced in planktonic culture have associated DNA (11, 12, 13, 20, 21, 30, 39, 48), could biofilm-derived MVs incorporate DNA (1, 39, 40, 44)?Open in a separate windowFIG. 1.Possible interactions between matrix polymers and particulate structures. Shown is an electron micrograph of a thin section through a P. aeruginosa PAO1 biofilm. During processing, some dehydration occurred, resulting in collapse of matrix material into fibrillate arrangements (black filled arrows). There is a suggestion of interactions occurring with particulate structures such as MVs (hollow white arrow) and flagella (filled white arrows) (identified by the appearance and cross-dimension of these highly ordered structures when viewed at high magnification), which was consistently observed with other embedded samples and also with whole-mount preparations of gently disrupted biofilms (data not shown). The scale bar represents 200 nm.     

Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

Open reading frame 45 (ORF45) of Kaposi''s sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.Kaposi''s sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus and the causative agent of several human cancers, including Kaposi''s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman''s disease (3, 6). Like all herpesviruses, KSHV has two alternative life cycles, a latent and a lytic cycle. During latency, only a few viral genes are expressed, and no progeny viruses are produced. Under appropriate conditions, latent viral genomes are activated, initiate lytic replication, and express a full panel of viral genes, in a process that leads to viral assembly, release of progeny virus particles, and de novo infection of naïve cells (3, 6). KSHV establishes latent infection in the majority of infected cells in cases of KS, primary effusion lymphoma, and multicentric Castleman''s disease, but lytic replications occur in a small fraction. The recurrent and periodic lytic cycles of KSHV are believed to play critical roles in viral pathogenesis (6, 7).Open reading frame 45 (ORF45) is a KSHV-encoded gene product that plays a critical role in the viral lytic cycle. It is an immediate-early protein and is also present in viral particles as tegument protein (26, 27, 30). Disruption of ORF45 has no significant effect on overall viral lytic gene expression or DNA replication in BAC36-reconstituted 293T cells induced with both tetradecanoyl phorbol acetate (TPA) and sodium butyrate together, but the ORF45-null mutant produces 5- to 10-fold fewer progeny viruses than the wild type and the mutant virus has dramatically reduced infectivity, suggesting that ORF45 plays important roles at both early and late stages of viral infection (29). In addition to its roles as a tegument component, which are possibly involved in viral ingress and egress processes, KSHV ORF45 interacts with cellular proteins and modulates the cellular environment. At least two such functions have been described. First, KSHV ORF45 inhibits activation of interferon regulatory factor 7 (IRF-7) and therefore antagonizes the host innate antiviral response (28). Second, KSHV ORF45 interacts with p90 ribosomal kinase 1 and 2 (RSK1/RSK2) and modulates the extracellular signal-regulated kinase/RSK signaling pathway, which is known to play essential roles in KSHV reactivation and lytic replication (12). All of these data suggest that KSHV ORF45 is a multifunctional protein.ORF45 is unique to the gammaherpesviruses; it has no homologue in the alpha- or betaherpesviruses. ORF45 homologues have been identified as virion protein components in other gammaherpesviruses, such as Epstein-Barr virus (EBV), rhesus rhadinovirus (RRV), and murine herpesvirus 68 (MHV-68), suggesting that certain tegument functions of ORF45 are conserved (2, 11, 18). ORF45 homologues differ in protein length. KSHV ORF45 is the longest, at 407 amino acids (aa); RRV, EBV, MHV-68, and herpesvirus saimiri (HVS) have proteins of 353, 217, 206, and 257 aa, respectively. The limited homologies lie mostly at the amino- and carboxyl-terminal ends. The middle portion of KSHV ORF45 diverges from those of its homologues. The homologues differ in subcellular localization. We and others have reported previously that KSHV ORF45 is found predominantly in the cytoplasm (1, 21, 28, 30), whereas ORF45 of MHV-68 is found exclusively in the nucleus (9). Recently, we found KSHV ORF45 also present in the nuclei of BCBL-1 cells in what resembled viral replication compartments, suggesting that ORF45 could shuttle into the nucleus (12).Nucleocytoplasmic trafficking of proteins across the nuclear membrane occurs through nuclear pore complexes. Small molecules of up to approximately 9 nm in diameter, corresponding to a globular protein of approximately 40 to 60 kDa, can in principle enter or leave the nucleus by diffusion through nuclear pores (15, 17, 24). Large molecules are transported with the aid of a related family of transport factors, importins and exportins, which recognize nuclear localization sequence (NLS)-containing or nuclear export sequence (NES)-containing proteins (15, 17, 23). CRM1 (exportin 1) has been identified as a common export receptor that recognizes human immunodeficiency virus Rev-like leucine-rich NES sequences and is responsible for the export of such NES-containing proteins (4, 5, 19, 22). CRM1-dependent nuclear export is specifically inhibited by a pharmacological compound, leptomycin B (LMB), that interacts with CRM1 and thus blocks such NES-mediated protein export (4).To understand the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we attempted to locate the signals that control its subcellular localization. In the research reported here, we identified a leucine-rich NES and an adjacent basic NLS in KSHV ORF45. We demonstrated that the regulated intracellular trafficking of ORF45, especially its translocation into the nucleus, is important for KSHV lytic replication.     

Gene Regulation and Functional Alterations Induced by Kaposi's Sarcoma-Associated Herpesvirus-Encoded ORFK13/vFLIP in Endothelial Cells

Kaposi''s sarcoma (KS) is an angioproliferative inflammatory disorder induced by endothelial cell infection with the KS-associated herpesvirus (KSHV). ORFK13/vFLIP, one of the KSHV genes expressed in KS, encodes a 188-amino-acid protein which binds to the Iκb kinase (IKK) complex to activate NF-κB. We examined ORFK13/vFLIP contribution to KS phenotype and potential for therapeutic targeting. Retroviral transduction of ORFK13/vFLIP into primary human endothelial cells induces the spindle morphology distinctive of KS cells and promotes the formation of abnormal vascular networks typical of KS vasculature; upregulates the expression of proinflammatory cytokines, chemokines, and interferon-responsive genes; and stimulates the adhesion of inflammatory cells characteristic of KS lesions. Thymidine phosphorylase, a cellular enzyme markedly induced by ORFK13/vFLIP, can metabolize the prodrug 5-fluoro-5-deoxyuridine (5-dFUrd) to 5-fluouridine (5-FU), a potent thymidine synthase inhibitor, which blocks DNA and RNA synthesis. When tested for cytotoxicity, 5-dFUrd (0.1 to 1 μM) selectively killed ORFK13/vFLIP-expressing endothelial cells while sparing control cells. These results demonstrate that ORFK13/vFLIP directly and indirectly contributes to the inflammatory and vascular phenotype of KS and identify 5-dFUrd as a potential new drug that targets KSHV latency for the treatment of KS and other KSHV-associated malignancies.Kaposi''s sarcoma-associated herpesvirus (KSHV/human herpesvirus 8) is the etiological agent of Kaposi''s sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman''s diseases. KS typically presents as a multicentric angioproliferative tumor characterized by multiple nodular or macular lesions often on the skin, and less frequently in the gastrointestinal tract and the lung. Histologically, the lesions consist of spindle cells infected with KSHV, inflammatory infiltrates of monocytes/macrophages, lymphocytes and other cells, and “vascular slits” replete of red blood cells (8). KS spindle cells are likely to be of endothelial lineage (19).In KS tissues, KSHV establishes a mostly latent infection characterized by expression of a limited number of viral genes that are likely important to the disease pathogenesis (30). ORFK13 is one such KSHV latent gene. Its gene product, called vFLIP (for viral Flice-like inhibitory protein) or K-FLIP, comprises two tandem death-effector domains that are often found in apoptotic signaling mediators such as cellular FLICE inhibitory protein (cFLIP) and caspase-8/FLICE. Consistent with its sequence similarity with cFLIP, vFLIP was found to inhibit caspase activation and prevent apoptotic cell death (39). Silencing ORFK13/vFLIP expression by RNA interference stopped PEL growth in vitro and in vivo, providing evidence of the essential role of K13/vFLIP in PEL pathogenesis (16). Transgenic mice of K13/vFLIP in lymphoid cells developed more lymphomas than controls (11). Similar to the viral proteins of many other lymphogenic viruses, K13/vFLIP activates NF-κB (1, 10, 25, 27, 42). By activating NF-κB and inhibiting the AP-1 pathway, K13/vFLIP was recently reported to promote viral latency (49).Recent studies have characterized selected effects of K13/vFLIP expression in primary endothelial cells transduced with ORFK13/vFLIP (15, 20, 29), providing important insights into its function. Here, we broadly investigated K13/vFLIP function in endothelial cells. By establishing stable retrovirus-mediated transduction of ORFK13/vFLIP in primary human endothelial cells, we have extensively characterized the biochemical and functional consequences of K13/vFLIP expression in these cells.     

Metabolic Engineering of Saccharomyces cerevisiae for Production of Novel Cyanophycins with an Extended Range of Constituent Amino Acids

Cyanophycin (multi-l-arginyl-poly-l-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a putative precursor for numerous biodegradable technically used chemicals. Therefore, the biosynthesis and production of the polymer in recombinant organisms is of special interest. The synthesis of cyanophycin derivatives consisting of a wider range of constituents would broaden the applications of this polymer. We applied recombinant Saccharomyces cerevisiae strains defective in arginine metabolism and expressing the cyanophycin synthetase of Synechocystis sp. strain PCC 6308 in order to synthesize CGP with citrulline and ornithine as constituents. Strains defective in arginine degradation (Car1 and Car2) accumulated up to 4% (wt/wt) CGP, whereas strains defective in arginine synthesis (Arg1, Arg3, and Arg4) accumulated up to 15.3% (wt/wt) of CGP, which is more than twofold higher than the previously content reported in yeast and the highest content ever reported in eukaryotes. Characterization of the isolated polymers by different analytical methods indicated that CGP synthesized by strain Arg1 (with argininosuccinate synthetase deleted) consisted of up to 20 mol% of citrulline, whereas CGP from strain Arg3 (with ornithine carbamoyltransferase deleted) consisted of up to 8 mol% of ornithine, and CGP isolated from strain Arg4 (with argininosuccinate lyase deleted) consisted of up to 16 mol% lysine. Cultivation experiments indicated that the incorporation of citrulline or ornithine is enhanced by the addition of low amounts of arginine (2 mM) and also by the addition of ornithine or citrulline (10 to 40 mM), respectively, to the medium.Cyanophycin (multi-l-arginyl-poly-[l-aspartic acid]), also referred to as cyanophycin grana peptide (CGP), represents a polydisperse nonribosomally synthesized polypeptide consisting of poly(aspartic acid) as backbone and arginine residues bound to each aspartate (49) (Fig. ​(Fig.1).1). One enzyme only, referred to as cyanophycin synthetase (CphA), catalyzes the synthesis of the polymer from amino acids (55). Several CphAs originating from different bacteria exhibit specific features (2, 7, 5, 32, 50, 51). CphAs from the cyanobacteria Synechocystis sp. strain PCC 6308 and Anabaena variabilis ATCC 29413, respectively, exhibit a wide substrate range in vitro (2, 7), whereas CphA from Acinetobacter baylyi or Nostoc ellipsosporum incorporates only aspartate and arginine (23, 24, 32). CphA from Thermosynechococcus elongatus catalyzes the synthesis of CGP primer independently (5); CphA from Synechococcus sp. strain MA19 exhibits high thermostability (22). Furthermore, two types of CGP were observed concerning its solubility behavior: (i) a water-insoluble type that becomes soluble at high or low pH (34, 48) and (ii) a water-soluble type that was only recently observed in recombinant organisms (19, 26, 42, 50, 56). In the past, bacteria were mainly applied for the synthesis of CGP (3, 14, 18, 53), whereas recently there has been greater interest in synthesis in eukaryotes (26, 42, 50). CGP was accumulated to almost 7% (wt/wt) of dry matter in recombinant Nicotiana tabacum and Saccharomyces cerevisiae (26, 50).Open in a separate windowFIG. 1.Chemical structures of dipeptide building blocks of CGP variants detected in vivo. Structure: 1, aspartate-arginine; 2, aspartate-lysine; 3, aspartate-citrulline; 4, aspartate-ornithine. Aspartic acid is presented in black; the second amino acid of the dipeptide building blocks is shown in gray. The nomenclature of the carbon atoms is given.In S. cerevisiae the arginine metabolism is well understood and has been investigated (30) (see Fig. ​Fig.2).2). Arginine is synthesized from glutamate via ornithine and citrulline in eight successive steps. The enzymes acetylglutamate synthase, acetylglutamate kinase, N-acetyl-γ-glutamylphosphate reductase, and acetylornithine aminotransferase are involved in the formation of N-α-acetylornithine. The latter is converted to ornithine by acetylornithine acetyltransferase. In the next step, ornithine carbamoyltransferase (ARG3) condenses ornithine with carbamoylphosphate, yielding citrulline. Citrulline is then converted to l-argininosuccinate by argininosuccinate synthetase. The latter is in the final step cleaved into fumarate and arginine by argininosuccinate lyase (ARG4). The first five steps occur in the mitochondria, whereas the last three reactions occur in the cytosol (28, 54). Arginine degradation is initiated by arginase (CAR1) and ornithine aminotransferase (CAR2) (10, 11, 38, 39).Open in a separate windowFIG. 2.Schematic overview of the arginine metabolism in S. cerevisiae. Reactions shown in the shaded area occur in the mitochondria, while the other reactions are catalyzed in the cytosol. Abbreviations: ARG2, acetylglutamate synthase; ARG6, acetylglutamate kinase; ARG5, N-acetyl-γ-glutamyl-phosphate reductase; ARG8, acetylornithine aminotransferase; ECM40, acetylornithine acetyltransferase; ARG1, argininosuccinate synthetase; ARG3, ornithine carbamoyltransferase; ARG4, argininosuccinate lyase; CAR1, arginase; CAR2, ornithine aminotransferase.A multitude of putative applications for CGP derivatives are available (29, 41, 45, 47), thus indicating a need for efficient biotechnological production and for further investigations concerning the synthesis of CGP with alternative properties and different constituents. It is not only the putative application of the polymer as a precursor for poly(aspartic acid), which is used as biodegradable alternative for poly(acrylic acid) or for bulk chemicals, that makes CGP interesting (29, 45-47). In addition, a recently developed process for the production of dipeptides from CGP as a precursor makes the synthesis of CGP variants worthwhile (43). Dipeptides play an important role in medicine and pharmacy, e.g., as additives for malnourished patients, as treatments against liver diseases, or as aids for muscle proliferation (43). Because dipeptides are synthesized chemically (40) or enzymatically (6), novel biotechnological production processes are welcome.     

Inhibition of the Activity of Both Domains of Lysostaphin through Peptidoglycan Modification by the Lysostaphin Immunity Protein

Resistance to lysostaphin, a staphylolytic glycylglycine endopeptidase, is due to a FemABX-like immunity protein that inserts serines in place of some glycines in peptidoglycan cross bridges. These modifications inhibit both binding of the recombinant cell wall targeting domain and catalysis by the recombinant catalytic domain of lysostaphin.Lysostaphin is a glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus (18) that lyses susceptible staphylococci by hydrolyzing the polyglycine cross bridges in their cell wall peptidoglycans (3). The lysostaphin gene sequence was independently determined in 1987 by two groups (8, 13). BLAST analysis (1) of mature lysostaphin revealed two domains: an N-terminal catalytic domain (CAT), which is a member of the M23 family of zinc metalloendopeptidases, and a C-terminal cell wall targeting domain (CWT), which is a member of the SH3b domain family (Fig. ​(Fig.11 A).Open in a separate windowFIG. 1.(A) Schematic diagram of mature lysostaphin, the recombinant catalytic domain (rCAT) (lysostaphin residues 1 to 148), and the recombinant cell wall targeting domain (rCWT) (lysostaphin residues 149 to 246). The numbers represent the beginning and end of the domains, and the solid boxes indicate the N-terminal His₆ tag of the recombinant proteins. (B) SDS-PAGE analysis of rCAT and rCWT purified by a nickel affinity column. Mobilities of molecular mass standards are given on the left side of the gel.The lysostaphin endopeptidase resistance gene (epr or lif) encodes a FemABX-like immunity protein that is located adjacent to the lysostaphin gene on the plasmid pACK1 in S. simulans bv. staphylolyticus (4, 7, 20). Members of the FemABX family of proteins are nonribosomal peptidyl transferases that are involved in the addition of cross bridge amino acids during peptidoglycan subunit synthesis in the cytoplasm (15). In S. simulans bv. staphylolyticus, the lysostaphin immunity protein inserts serines in place of some glycines during peptidoglycan synthesis, which provides resistance to lysostaphin (4, 20).Originally it was suggested that the incorporation of serines in these peptidoglycan cross bridges gave increased resistance to lysostaphin because of the inability of the enzyme to hydrolyze glycyl-serine or seryl-glycine bonds (4, 14, 16). Others later reported that the CWT specifically binds to the polyglycine cross bridges in staphylococci (6) and the binding of CWT to producer-strain cells was less than that to susceptible cells (2). However, the ability of the enzyme or its targeting domain to bind to purified peptidoglycans from staphylococci containing the lysostaphin resistance gene has not been determined. Therefore, we determined if the modification to staphylococcal peptidoglycan cross bridges made by the lysostaphin immunity protein affected the activity of the binding domain, the catalytic domain, or both.     

Identification of the Biosynthetic Gene Cluster for the Pseudomonas aeruginosa Antimetabolite l-2-Amino-4-Methoxy-trans-3-Butenoic Acid

l-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of human infections and is considered the main pathogen responsible for chronic pneumonia in cystic fibrosis patients (7, 23). P. aeruginosa also infects other organisms, such as insects (4), nematodes (6), plants (18), and amoebae (20). Its ability to thrive as a pathogen and to compete in aquatic and soil environments can be partly attributed to the production and interplay of secreted virulence factors and secondary metabolites. While the importance of many of these exoproducts has been studied, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid (AMB; methoxyvinylglycine) (Fig. ​(Fig.1)1) has received only limited attention. Identified during a search for new antibiotics, AMB was found to reversibly inhibit the growth of Bacillus spp. (26) and Escherichia coli (25) and was later shown to inhibit the growth and metabolism of cultured Walker carcinosarcoma cells (28). AMB is a γ-substituted vinylglycine, a naturally occurring amino acid with a β,γ-C=C double bond. Other members of this family are aminoethoxyvinylglycine from Streptomyces spp. (19) and rhizobitoxine, made by Bradyrhizobium japonicum (16) and Pseudomonas andropogonis (15) (Fig. ​(Fig.1).1). As inhibitors of pyridoxal phosphate-dependent enzymes (13, 17, 21, 22), γ-substituted vinylglycines have multiple targets in bacteria, animals, and plants (3, 5, 10, 21, 22, 29). However, the importance of AMB as a toxin in biological interactions with P. aeruginosa has not been addressed, as AMB biosynthesis and the genes involved have not been elucidated.Open in a separate windowFIG. 1.Chemical structures of the γ-substituted vinylglycines AMB, aminoethoxyvinylglycine, and rhizobitoxine.     

Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum

In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.With an annual market volume of more than 1,000,000 tons, lysine is one of the dominating products in biotechnology. In recent years, rational metabolic engineering has emerged as a powerful tool for lysine production (16, 18, 22). Hereby, different target enzymes and pathways in the central metabolism could be identified and successfully modified to create superior production strains (1, 2, 5, 8, 10, 17-20). The tricarboxylic acid (TCA) cycle has not been rationally engineered so far, despite its major role in Corynebacterium glutamicum (6). From metabolic flux studies, however, it seems that the TCA cycle might offer a great potential for optimization (Fig. ​(Fig.1),1), which is also predicted from in silico pathway analysis (13, 22). Experimental evidence comes from studies with Brevibacterium flavum exhibiting increased lysine production due to an induced bottleneck toward the TCA cycle (21). In the present work, we performed TCA cycle engineering by downregulation of isocitrate dehydrogenase (ICD). ICD is the highest expressed TCA cycle enzyme in C. glutamicum (7). Downregulation was achieved by start codon exchange, controlling ICD expression on the level of translation.Open in a separate windowFIG. 1.Stoichiometric correlation of lysine yield (%), biomass yield (g/mol) and TCA cycle flux (%; entry flux through citrate synthase) determined by ¹³C metabolic flux analysis achieved by paraboloid fitting of the data set (parameters were determined with Y0 = 105.1, a = −1.27, b = 0.35, c = −9.35 × 10⁻³, d = −11.16 × 10⁻³). The data displayed represent values from 18 independent experiments with different C. glutamicum strains taken from previous studies (1-3, 11, 12, 15, 23).