Study of peripheral domains in structure-function of isocitrate lyase (ICL) from Pseudomonas aeruginosa.

The capacity of Pseudomonas aeruginosa to assimilate nutrients is essential for niche colonization and contributes to its pathogenicity. Isocitrate lyase (ICL), the first enzyme of the glyoxylate cycle, redirects isocitrate from the tricarboxylic acid cycle to render glyoxylate and succinate. P. aeruginosa ICL (PaICL) is regarded as a virulence factor due to its role in carbon assimilation during infection. The AceA/ICL protein family shares the catalytic domain I, triosephosphate isomerase barrel (TIM-barrel). The carboxyl terminus of domain I is essential for Escherichia coli ICL (EcICL) of subfamily 1. PaICL, which belongs to subfamily 3, has domain II inserted at the periphery of domain I, which is believed to participate in enzyme oligomerization. In addition, PaICL has the α13-loop-α14 (extended motif), which protrudes from the enzyme core, being of unknown function. This study investigates the role of domain II, the extended motif, and the carboxyl-terminus (C-ICL) and amino-terminus (N-ICL) regions in the function of the PaICL enzyme, also as their involvement in the virulence of P. aeruginosa PAO1. Deletion of domain II and the extended motif results in enzyme inactivation and structural instability of the enzyme. The His6-tag fusion at the C-ICL protein produced a less efficient enzyme than fusion at the N-ICL, but without affecting the acetate assimilation or virulence. The PaICL homotetrameric structure of the enzyme was more stable in the N-His6-ICL than in the C-His6-ICL, suggesting that the C-terminus is critical for the ICL quaternary conformation. The ICL-mutant A39 complemented with the recombinant proteins N-His6-ICL or C-His6-ICL were more virulent than the WT PAO1 strain. The findings indicate that the domain II and the extended motif are essential for the ICL structure/function, and the C-terminus is involved in its quaternary structure conformation, confirming that in P. aeruginosa, the ICL is essential for acetate assimilation and virulence.

Sodium and lithium exert differential effects on the central carbon metabolism of Debaryomyces hansenii through the glyoxylate shunt regulation.

Debaryomyces hansenii is a halotolerant/halophilic yeast usually found in salty environments. The yeast accumulated sodium at high concentrations, which improved growth in salty media. In contrast, lithium was toxic even at low concentrations and its presence prevented cell proliferation. To analyse the responses to both cations, metabolite levels, enzymatic activities and gene expression were determined, showing that NaCl and LiCl trigger different cellular responses. At high concentrations of NaCl (0.5 or 1.5 M) cells accumulated higher amounts of the intermediate metabolites glyoxylate and malate and, at the same time, the levels of intracellular oxoglutarate decreased. Additionally, 0.5 M NaCl increased the activity of the enzymes isocitrate lyase and malate synthase involved in the synthesis of glyoxylate and malate respectively and decreased the activity of isocitrate dehydrogenase. Moreover, transcription of the genes coding for isocitrate lyase and malate synthase was activated by NaCl. Also, cells accumulated phosphate upon NaCl exposure. None of these effects was provoked when LiCl (0.1 or 0.3 M) was used instead of NaCl. Lithium induced accumulation of higher amounts of oxoglutarate and decreased the concentrations of glyoxylate and malate to non-detectable levels. Cells incubated with lithium also showed higher activity of the isocitrate dehydrogenase and neither increased isocitrate lyase and malate synthase activities nor the transcription of the corresponding genes. In summary, we show that sodium, but not lithium, up regulates the shunt of the glyoxylic acid in D. hansenii and we propose that this is an important metabolic adaptation to thrive in salty environments.

Mycobacterium tuberculosis Rv1916 is an Acetyl-CoA-Binding Protein.

Isocitrate lyase (ICL) isoform 2 is an essential enzyme for some clinical Mycobacterium tuberculosis (Mtb) strains during infection. In the laboratory Mtb strain H37Rv, the icl2 gene encodes two distinct gene products - Rv1915 and Rv1916 - due to a frameshift mutation. This study aims to characterise these two gene products to understand their structure and function. While we were unable to produce Rv1915 recombinantly, soluble Rv1916 was obtained with sufficient yield for characterisation. Kinetic studies using UV-visible spectrophotometry and 1 H-NMR spectroscopy showed that recombinant Rv1916 does not possess isocitrate lyase activity, while waterLOGSY binding experiments demonstrated that it could bind acetyl-CoA. Finally, X-ray crystallography revealed structural similarities between Rv1916 and the C-terminal domain of ICL2. Considering the probable differences between full-length ICL2 and the gene products Rv1915 and Rv1916, care must be taken when using Mtb H37Rv as a model organism to study central carbon metabolism.

Characterization of a salt-resistant isocitrate lyase gene from mangrove wetland using shotgun metagenomic sequencing.

Isocitrate lyase (ICL), as the key enzyme in the glyoxylate metabolic pathway, plays an important role in metabolic adaptation to environmental changes. In this study, metagenomic DNA from the soil and water microorganism collected from the Dongzhai Harbor Mangroves (DHM) reserve, in Haikou City, China, was high-throughput sequenced using an Illumina HiSeq 4000 platform. The icl121 gene, encoding an ICL with the highly conserved catalytic pattern IENQVSDEKQCGHQD was identified. Then, this gene was subcloned into the pET-30a vector and overexpressed in Escherichia coli BL21 (DE3) cells. The maximum enzymatic activity of the recombinant ICL121 protein is 9.47 × 102 U/mg occurring at pH 7.5 and 37°C. Furthermore, as a metalo-enzyme, ICL121 can utilize the appropriate concentrations of Mg2+, Mn2+, and Na+ ion as cofactors to exhibit high enzymatic activity. In particular, the novel metagenomic-derived icl121 gene displayed distinct salt tolerance (NaCl) and might be useful for generating salt-tolerant crops in the future.

The Transcription Factor StuA Regulates the Glyoxylate Cycle in the Dermatophyte Trichophyton rubrum under Carbon Starvation.

Trichophyton rubrum is the primary causative agent of dermatophytosis worldwide. This fungus colonizes keratinized tissues and uses keratin as a nutritional source during infection. In T. rubrum-host interactions, sensing a hostile environment triggers the adaptation of its metabolic machinery to ensure its survival. The glyoxylate cycle has emerged as an alternative metabolic pathway when glucose availability is limited; this enables the conversion of simple carbon compounds into glucose via gluconeogenesis. In this study, we investigated the impact of stuA deletion on the response of glyoxylate cycle enzymes during fungal growth under varying culture conditions in conjunction with post-transcriptional regulation through alternative splicing of the genes encoding these enzymes. We revealed that the ΔstuA mutant downregulated the malate synthase and isocitrate lyase genes in a keratin-containing medium or when co-cultured with human keratinocytes. Alternative splicing of an isocitrate lyase gene yielded a new isoform. Enzymatic activity assays showed specific instances where isocitrate lyase and malate synthase activities were affected in the mutant strain compared to the wild type strain. Taken together, our results indicate a relevant balance in transcriptional regulation that has distinct effects on the enzymatic activities of malate synthase and isocitrate lyase.

Lessons Learnt and the Way Forward for Drug Development Against Isocitrate Lyase from Mycobacterium tuberculosis.

Isocitrate lyase (ICL), an enzyme of the glyoxylate shunt pathway, is essential for the virulence and persistence of dreaded Mycobacterium tuberculosis (Mtb) in its host. This pathway, along with the methylcitrate cycle, facilitates the utilization of fatty acids as a carbon source inside hostile host environments such as in granulomas, and hence enzymes of this pathway are novel antitubercular targets. The genome sequence of pathogenic Mtb H37Rv presents three ICLs annotated as Rv0467 (prokaryotic homologue), Rv1915 and Rv1916. The latter two, Rv1915 and Rv1916, together constitute the longer version of ICL2, a eukaryotic counterpart. Despite being a well-known drug target, no Mtb ICL inhibitor has reached clinical trials due to challenges associated with targeting all the 3 orthologs. This gap is the result of uncharacterized Rv1915 and Rv1916. This review aims to appreciate chronologically the key studies that have built our comprehension of Mtb ICLs. Recently characterized Mtb Rv1915 and Rv1916, which further open venues for developing effective inhibitors against the persistent and drug-resistant Mtb, are discussed separately.

Malate synthase contributes to the survival of Salmonella Typhimurium against nutrient and oxidative stress conditions.

To survive and replicate in the host, S. Typhimurium have evolved several metabolic pathways. The glyoxylate shunt is one such pathway that can utilize acetate for the synthesis of glucose and other biomolecules. This pathway is a bypass of the TCA cycle in which CO2 generating steps are omitted. Two enzymes involved in the glyoxylate cycle are isocitrate lyase (ICL) and malate synthase (MS). We determined the contribution of MS in the survival of S. Typhimurium under carbon limiting and oxidative stress conditions. The ms gene deletion strain (∆ms strain) grew normally in LB media but failed to grow in M9 minimal media supplemented with acetate as a sole carbon source. However, the ∆ms strain showed hypersensitivity (p < 0.05) to hypochlorite. Further, ∆ms strain has been significantly more susceptible to neutrophils. Interestingly, several folds induction of ms gene was observed following incubation of S. Typhimurium with neutrophils. Further, ∆ms strain showed defective colonization in poultry spleen and liver. In short, our data demonstrate that the MS contributes to the virulence of S. Typhimurium by aiding its survival under carbon starvation and oxidative stress conditions.

Rv1915 and Rv1916 from Mycobacterium tuberculosis H37Rv form in vitro protein-protein complex.

BACKGROUND: Mycobacterium tuberculosis (Mtb) isocitrate lyase (ICL) is an established drug target that facilitates Mtb persistence. Unlike other mycobacterial strains, where ICL2 is a single gene product, H37Rv has a split event, resulting in two tandemly coded icls - rv1915 and rv1916. Our recent report on functionality of individual Rv1915 and Rv1916, led to postulate the cooperative role of these proteins in pathogen's survival under nutrient-limiting conditions. This study investigates the possibility of Rv1915 and Rv1916 interacting and forming a complex. METHODS: Pull down assay, activity assay, mass spectrometry and site directed mutagenesis was employed to investigate and validate Rv1915-Rv1916 complex formation. RESULTS: Rv1915 and Rv1916 form a stable complex in vitro, with enhanced ICL/MICL activities as opposed to individual proteins. Further, activities monitored in the presence of acetyl-CoA show significant increase for Rv1916 and the complex but not of Rv0467 and Rv1915Δ90CT. Both full length and truncated Rv1915Δ90CT can form complex, implying the absence of its C-terminal disordered region in complex formation. Further, in silico analysis and site-directed mutagenesis studies reveal Y64 and Y65 to be crucial residues for Rv1915-Rv1916 complex formation. CONCLUSIONS: This study uncovers the association between Rv1915 and Rv1916 and supports the role of acetyl-CoA in escalating the ICL/MICL activities of Rv1916 and Rv1915Δ90CT-Rv1916 complex. GENERAL SIGNIFICANCE: Partitioning of ICL2 into Rv1915 and Rv1916 that associates to form a complex in Mtb H37Rv, suggests its importance in signaling and regulation of metabolic pathway particularly in carbon assimilation.

Regulation of the icl1 Gene Encoding the Major Isocitrate Lyase in Mycobacterium smegmatis.

Mycobacterium smegmatis has two isocitrate lyase (ICL) isozymes (MSMEG_0911 and MSMEG_3706). We demonstrated that ICL1 (MSMEG_0911) is the predominantly expressed ICL in M. smegmatis and plays a major role in growth on acetate or fatty acid as the sole carbon and energy source. Expression of the icl1 gene in M. smegmatis was demonstrated to be strongly upregulated during growth on acetate relative to that in M. smegmatis grown on glucose. Expression of icl1 was shown to be positively regulated by the RamB activator, and three RamB-binding sites (RamBS1, RamBS2, and RamBS3) were identified in the upstream region of icl1 using DNase I footprinting analysis. Succinyl coenzyme A (succinyl-CoA) was shown to increase the affinity of binding of RamB to its binding sites and enable RamB to bind to RamBS2, which is the most important site for RamB-mediated induction of icl1 expression. These results suggest that succinyl-CoA serves as a coinducer molecule for RamB. Our study also showed that cAMP receptor protein (Crp1; MSMEG_6189) represses icl1 expression in M. smegmatis grown in the presence of glucose. Therefore, the strong induction of icl1 expression during growth on acetate as the sole carbon source relative to the weak expression of icl1 during growth on glucose is likely to result from combined effects of RamB-mediated induction of icl1 in the presence of acetate and Crp-mediated repression of icl1 in the presence of glucose. IMPORTANCE Carbon flux through the glyoxylate shunt has been suggested to affect virulence, persistence, and antibiotic resistance of Mycobacterium tuberculosis. Therefore, it is important to understand the precise mechanism underlying the regulation of the icl gene encoding the key enzyme of the glyoxylate shunt. Using Mycobacterium smegmatis, this study revealed the regulation mechanism underlying induction of icl1 expression in M. smegmatis when the glyoxylate shunt is required. The conservation of the cis- and trans-acting regulatory elements related to icl1 regulation in both M. smegmatis and M. tuberculosis implies that a similar regulatory mechanism operates for the regulation of icl1 expression in M. tuberculosis.

Inhibitory Effects of Epipolythiodioxopiperazine Fungal Metabolites on Isocitrate Lyase in the Glyoxylate Cycle of Candida albicans.

Four epipolythiodioxopiperazine fungal metabolites (1-4) isolated from the sponge-derived Aspergillus quadrilineatus FJJ093 were evaluated for their capacity to inhibit isocitrate lyase (ICL) in the glyoxylate cycle of Candida albicans. The structures of these compounds were elucidated using spectroscopic techniques and comparisons with previously reported data. We found secoemestrin C (1) (an epitetrathiodioxopiperazine derivative) to be a potent ICL inhibitor, with an inhibitory concentration of 4.77 ± 0.08 µM. Phenotypic analyses of ICL-deletion mutants via growth assays with acetate as the sole carbon source demonstrated that secoemestrin C (1) inhibited C. albicans ICL. Semi-quantitative reverse-transcription polymerase chain reaction analyses indicated that secoemestrin C (1) inhibits ICL mRNA expression in C. albicans under C2-assimilating conditions.

Structural insights into the targeting specificity of ubiquitin ligase for S. cerevisiae isocitrate lyase but not C. albicans isocitrate lyase.

In Saccharomyces cerevisiae, the glyoxylate cycle is controlled through the posttranslational regulation of its component enzymes, such as isocitrate lyase (ICL), which catalyzes the first unique step of the cycle. The ICL of S.cerevisiae (ScIcl1) is tagged for proteasomal degradation through ubiquitination by a multisubunit ubiquitin ligase (the glucose-induced degradation-deficient (GID) complex), whereas that of the pathogenic yeast Candida albicans (CaIcl1) escapes this process. However, the reason for the ubiquitin targeting specificity of the GID complex for ScIcl1 and not for CaIcl1 is unclear. To gain some insight into this, in this study, the crystal structures of apo ScIcl1 and CaIcl1 in complex with formate and the cryogenic electron microscopy structure of apo CaIcl1 were determined at a resolution of 2.3, 2.7, and 2.6 Å, respectively. A comparison of the various structures suggests that the orientation of N-terminal helix α1 in S.cerevisiae is likely key to repositioning of ubiquitination sites and contributes to the distinction found in C. albicans ubiquitin evasion mechanism. This finding gives us a better understanding of the molecular mechanism of ubiquitin-dependent ScIcl1 degradation and could serve as a theoretical basis for the research and development of anti-C. albicans drugs based on the concept of CaIcl1 ubiquitination.

RegX3 Controls Glyoxylate Shunt and Mycobacteria Survival by Directly Regulating the Transcription of Isocitrate Lyase Gene in Mycobacterium smegmatis.

The glyoxylate shunt is a pathway associated with the assimilation of fatty acids and is implicated in the resistance of M. tuberculosis (Mtb). Isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, mediates Mtb infections and its survival in mice via fatty acids, metabolism, and physiological functions. Here, we found that in Mycobacterium smegmatis (M. smegmatis) the two-component system SenX3-RegX3 regulated the glyoxylate shunt in response to phosphate starvation by controlling the transcription of icl. In response to phosphate availability, the phosphate regulator RegX3 directly bound to the upstream regulatory region of icl and repressed its transcription. The inactivation of regX3 increased icl transcription and ICL activity, causing a growth defect in M. smegmatis with fatty acids as the sole source of carbon and energy. The growth defect was partly due to the toxicity of the excess glyoxylate produced by ICL. A decrease in glyoxylic acid levels, overexpression of regX3, or the chemical inhibition (IA or 3-NP) of ICL restored the growth of the Regx3-deficient M. smegmatis. Thus, we established a genetic network between the phosphate stress response and glyoxylate shunt based on the amount of intracellular ICL during mycobacterial survival on short-chain fatty acids, which contributed to its antimicrobial arsenal.

A Set of Active Promoters with Different Activity Profiles for Superexpressing Rhodococcus Strain.

Rhodococcus bacteria are a promising platform for biodegradation, biocatalysis, and biosynthesis, but the use of rhodococci is hampered by the insufficient number of both platform strains for expression and promoters that are functional and thoroughly studied in these strains. To expand the list of such strains and promoters, we studied the expression capability of the Rhodococcus rhodochrous M33 strain, and the functioning of a set of recombinant promoters in it. We showed that the strain supports superexpression of the target enzyme (nitrile hydratase) using alternative inexpensive feedings-acetate and urea-without growth factor supplementation, thus being a suitable expression platform. The promoter set included Ptuf (elongation factor Tu) and Psod (superoxide dismutase) from Corynebacterium glutamicum ATCC13032, Pcpi (isocitrate lyase) from Rhodococcus erythropolis PR4, and Pnh (nitrile hydratase) from R. rhodochrous M8. Activity levels, regulation possibilities, and growth-phase-dependent activity profiles of these promoters were studied in derivatives of the M33 strain. The activities of the promoters were significantly different (Pcpi < Psod ≪ Ptuf < Pnh), covering 103-fold range, and the most active Pnh and Ptuf produced up to a 30-50% portion of target protein in soluble intracellular proteins. On the basis of the mRNA quantification and amount of target protein, the production level of Pnh was positioned close to the theoretical upper limit of expression in a bacterial cell. A selection method for the laboratory evolution of such active promoters directly in Rhodococcus was also proposed. Concerning regulation, Ptuf could not be regulated (2-fold change), while others were tunable (6-fold for Psod, 79-fold for Pnh, and 44-fold for Pcpi). The promoters possessed four different activity profiles, including three with peak of activity at different growth phases and one with constant activity throughout the growth phases. Ptuf and Pcpi did not change their activity profile under different growth conditions, whereas the Psod and Pnh profiles changed depending on the growth media. The results allow flexible construction of Rhodococcus strains using the studied promoters, and demonstrate a valuable approach for complex characterization of promoters intended for biotechnological strain construction.

Botrytis cinerea methyl isocitrate lyase mediates oxidative stress tolerance and programmed cell death by modulating cellular succinate levels.

Fungi lack the entire animal core apoptotic machinery. Nevertheless, regulated cell death with apoptotic markers occurs in multicellular as well as in unicellular fungi and is essential for proper fungal development and stress adaptation. The discrepancy between appearance of an apoptotic-like regulated cell death (RCD) in the absence of core apoptotic machinery is further complicated by the fact that heterologous expression of animal apoptotic genes in fungi affects fungal RCD. Here we describe the role of BcMcl, a methyl isocitrate lyase from the plant pathogenic fungus Botrytis cinerea, in succinate metabolism, and the connection of succinate with stress responses and cell death. Over expression of bcmcl resulted in elevated tolerance to oxidative stress and reduced levels of RCD, which were associated with accumulation of elevated levels of succinate. Deletion of bcmcl had almost no effect on fungal development or stress sensitivity, and succinate levels were unchanged in the deletion strain. Gene expression experiments showed co-regulation of bcmcl and bcicl (isocitrate lyase); expression of the bcicl gene was enhanced in bcmcl deletion and suppressed in bcmcl over expression strains. External addition of succinate reproduced the phenotypes of the bcmcl over expression strains, including developmental defects, reduced virulence, and improved oxidative stress tolerance. Collectively, our results implicate mitochondria metabolic pathways, and in particular succinate metabolism, in regulation of fungal stress tolerance, and highlight the role of this onco-metabolite as potential mediator of fungal RCD.

Genome Scale Metabolic Model of the versatile methanotroph Methylocella silvestris.

BACKGROUND: Methylocella silvestris is a facultative aerobic methanotrophic bacterium which uses not only methane, but also other alkanes such as ethane and propane, as carbon and energy sources. Its high metabolic versatility, together with the availability of tools for its genetic engineering, make it a very promising platform for metabolic engineering and industrial biotechnology using natural gas as substrate. RESULTS: The first Genome Scale Metabolic Model for M. silvestris is presented. The model has been used to predict the ability of M. silvestris to grow on 12 different substrates, the growth phenotype of two deletion mutants (ΔICL and ΔMS), and biomass yield on methane and ethanol. The model, together with phenotypic characterization of the deletion mutants, revealed that M. silvestris uses the glyoxylate shuttle for the assimilation of C1 and C2 substrates, which is unique in contrast to published reports of other methanotrophs. Two alternative pathways for propane metabolism have been identified and validated experimentally using enzyme activity tests and constructing a deletion mutant (Δ1641), which enabled the identification of acetol as one of the intermediates of propane assimilation via 2-propanol. The model was also used to integrate proteomic data and to identify key enzymes responsible for the adaptation of M. silvestris to different substrates. CONCLUSIONS: The model has been used to elucidate key metabolic features of M. silvestris, such as its use of the glyoxylate shuttle for the assimilation of one and two carbon compounds and the existence of two parallel metabolic pathways for propane assimilation. This model, together with the fact that tools for its genetic engineering already exist, paves the way for the use of M. silvestris as a platform for metabolic engineering and industrial exploitation of methanotrophs.

Salmonella enterica's "Choice": Itaconic Acid Degradation or Bacteriocin Immunity Genes.

Itaconic acid is an immunoregulatory metabolite produced by macrophages in response to pathogen invasion. It also exhibits antibacterial activity because it is an uncompetitive inhibitor of isocitrate lyase, whose activity is required for the glyoxylate shunt to be operational. Some bacteria, such as Yersinia pestis, encode enzymes that can degrade itaconic acid and therefore eliminate this metabolic inhibitor. Studies, primarily with Salmonella enterica subspecies enterica serovar Typhimurium, have demonstrated the presence of similar genes in this pathogen and the importance of these genes for the persistence of the pathogen in murine hosts. This minireview demonstrates that, based on Blast searches of 1063 complete Salmonella genome sequences, not all Salmonella serovars possess these genes. It is also shown that the growth of Salmonella isolates that do not possess these genes is sensitive to the acid under glucose-limiting conditions. Interestingly, most of the serovars without the three genes, including serovar Typhi, harbor DNA at the corresponding genomic location that encodes two open reading frames that are similar to bacteriocin immunity genes. It is hypothesized that these genes could be important for Salmonella that finds itself in strong competition with other Enterobacteriacea in the intestinal tract-for example, during inflammation.

Transcriptional regulation of central carbon metabolism in Pseudomonas aeruginosa.

Microbes such as Pseudomonas aeruginosa are often challenged by rapidly changing nutritional environments. In order to adapt to these shifts in nutrient availability, bacteria exert tight transcriptional control over the enzymes of central metabolism. This transcriptional control is orchestrated by a series of transcriptional repressors and activators. Although a number of these transcription factors have been identified, many others remain uncharacterized. Here, we present a simple pipeline to uncover and validate the targets of uncharacterized transcriptional regulators in P. aeruginosa. We use this approach to identify and confirm that an orthologue of the Pseudomonas fluorescens transcriptional regulator (RccR) binds to the upstream region of isocitrate lyase (aceA) in P. aeruginosa, thereby repressing flux through the glyoxylate shunt during growth on non-C2 carbon sources.

Recognition of gluconeogenic enzymes; Icl1, Fbp1, and Mdh2 by Gid4 ligase: A molecular docking study.

The pro/N-degron pathway is an evolved protein degradation pathway through the ubiquitin-proteasome system. It is a vital pathway to attain protein homeostasis inside the liver cells with varying glucose levels. N-terminal proline exists in more than 300 proteins in Saccharomyces cerevisiae, but only three of them are the gluconeogenic enzymes; isocitrate lyase (Icl1), fructose-1,6-bisphosphatase (Fbp1), and malate dehydrogenase (Mdh2). The present in silico study aims to structurally illustrate the binding of Icl1 enzyme to Gid4 ligase concerning its peers; Fbp1 and Mdh2. Based on the molecular docking scores and interactions, one can attribute the binding stability of Gid4 with degrons, to peptides of length six up to eight from the N-terminal. Moreover, the percent change in the docking score provides a rationale for the unique Gid4-Icl11-4 interaction. The present study provides insights on the binding attitude of Gid4 ligase to degrons of different lengths, so one will consider in designing peptidomimetics to target Gid4 ligase.

Isocitrate lyase plays important roles in plant salt tolerance.

BACKGROUND: Isocitrate lyase (ICL) is a key enzyme in the glyoxylate cycle. In a previous study in rice, the expression of the ICL-encoding gene (OsICL) was highly induced by salt stress and its expression was enhanced in transgenic rice lines overexpressing OsCam1-1, a calmodulin (CaM)-encoding gene. CaM has been implicated in salt tolerance mechanisms in plants; however, the cellular mechanisms mediated by CaM are not clearly understood. In this study, the role of OsICL in plant salt tolerance mechanisms and the possible involvement of CaM were investigated using transgenic plants expressing OsICL or OsCam1-1. RESULTS: OsICL was highly expressed in senesced leaf and significantly induced by salt stress in three OsCam1-1 overexpressing transgenic rice lines as well as in wild type (WT). In WT young leaf, although OsICL expression was not affected by salt stress, all three transgenic lines exhibited highly induced expression levels. In Arabidopsis, salt stress had negative effects on germination and seedling growth of the AtICL knockout mutant (Aticl mutant). To examine the roles of OsICL we generated the following transgenic Arabidopsis lines: the Aticl mutant expressing OsICL driven by the native AtICL promoter, the Aticl mutant overexpressing OsICL driven by the 35SCaMV promoter, and WT overexpressing OsICL driven by the 35SCaMV promoter. Under salt stress, the germination rate and seedling fresh and dry weights of the OsICL-expressing lines were higher than those of the Aticl mutant, and the two lines with the icl mutant background were similar to the WT. The Fv/Fm and temperature of rosette leaves in the OsICL-expressing lines were less affected by salt stress than they were in the Aticl mutant. Finally, glucose and fructose contents of the Aticl mutant under salt stress were highest, whereas those of OsICL-expressing lines were similar to or lower than those of the WT. CONCLUSIONS: OsICL, a salt-responsive gene, was characterized in the transgenic Arabidopsis lines, revealing that OsICL expression could revert the salt sensitivity phenotypes of the Aticl knockout mutant. This work provides novel evidence that supports the role of ICL in plant salt tolerance through the glyoxylate cycle and the possible involvement of OsCam1-1 in regulating its transcription.

Alternative fate of glyoxylate during acetate and hexadecane metabolism in Acinetobacter oleivorans DR1.

The glyoxylate shunt (GS), involving isocitrate lyase (encoded by aceA) and malate synthase G (encoded by glcB), is known to play important roles under several conditions including oxidative stress, antibiotic defense, or certain carbon source metabolism (acetate and fatty acids). Comparative growth analyses of wild type (WT), aceA, and glcB null-strains revealed that aceA, but not glcB, is essential for cells to grow on either acetate (1%) or hexadecane (1%) in Acinetobacter oleivorans DR1. Interestingly. the aceA knockout strain was able to grow slower in 0.1% acetate than the parent strain. Northern Blot analysis showed that the expression of aceA was dependent on the concentration of acetate or H2O2, while glcB was constitutively expressed. Up-regulation of stress response-related genes and down-regulation of main carbon metabolism-participating genes in a ΔaceA mutant, compared to that in the parent strain, suggested that an ΔaceA mutant is susceptible to acetate toxicity, but grows slowly in 0.1% acetate. However, a ΔglcB mutant showed no growth defect in acetate or hexadecane and no susceptibility to H2O2, suggesting the presence of an alternative pathway to eliminate glyoxylate toxicity. A lactate dehydrogenase (LDH, encoded by a ldh) could possibly mediate the conversion from glyoxylate to oxalate based on our RNA-seq profiles. Oxalate production during hexadecane degradation and impaired growth of a ΔldhΔglcB double mutant in both acetate and hexadecane-supplemented media suggested that LDH is a potential detoxifying enzyme for glyoxylate. Our constructed LDH-overexpressing Escherichia coli strain also showed an important role of LDH under lactate, acetate, and glyoxylate metabolisms. The LDH-overexpressing E. coli strain, but not wild type strain, produced oxalate under glyoxylate condition. In conclusion, the GS is a main player, but alternative glyoxylate pathways exist during acetate and hexadecane metabolism in A. oleivorans DR1.