Structural analysis of genes participating in melibiose fermentation and isocitrate lyase production in Yersinia pestis strains of main and non main subspecies

Comparative analysis of nucleotide sequences of genes participating in melibiose fermentation and isocitrate lyase production was conducted in 90 natural Yersinia pestis strains of main and non main subspecies. It was ascertained that the lack of the ability to utilize disaccharide melibiose in strains of the main subspecies is caused by integration of the insertion sequence IS285 at 73 bp from the beginning of the structural gene melB that encodes the transport protein galactoside permease. In contrast, strains of non main subspecies (caucasica, altaica, and ulegeica) contain the intact gene melB and are capable of fermenting melibiose. Differences in the manifestation of the other differential trait, production of isocitrate lyase, are connected with the presence of mutation (insertion of two nucleotides +CC) in the regulatory gene iclR encoding repressor protein of the acetate operon, which is the reason for constitutive synthesis of this enzyme. Strains of non main subspecies do not contain mutations in gene iclR, and this correlates in these strains with their capacity for inducible synthesis of isocitrate lyase.     

Sequence analysis of the yadA, inv, and ail genes and their expression in the main and nonmain Yersinia pestis subspecies and Yersinia pseudotuberculosis

The nucleotide sequences of the inv, yadA, and ail adhesin-invasin genes were analyzed in 24 strains of the main and nonmain Yersinia pestis subspecies, which were isolated from natural plague foci in Russia and neighbor countries, and ten Y. pseudotuberculosis strains. All of the five plague agent subspecies (main, caucasica, altaica, ulegeica, and hissarica) had the inv and yadA genes altered by insertion of the IS element and a single nucleotide deletion, respectively, as was earlier observed for the Y. pestis strains KIM and CO92. Consequently, the strains lacked functional activity of the Inv and YadA proteins. The ail gene of the main and ulegeica subspecies had a missense mutation, which replaced Val138 with Phe in the Ail protein. The strains of the caucasica subspecies had an AGT insertion in the ail gene, resulting in Ser148 insertion in the polypeptide chain. The changes in the ail sequence probably exerted no effect on ail expression, since the strains of all subspecies were resistant to blood serum complement.     

Genetic bases of methionine dependence in <Emphasis Type="Italic">Yersinia pestis</Emphasis> strains of main and non-main subspecies

Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and non-main subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and non-main subspecies revealed that the reason for the methionine dependence of strains belonging to the main subspecies is a deletion of a single nucleotide (−G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis.     

Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans

     The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creA ^d -30 strain showed that the creA gene is not involved in this process. Received: 13 May 1994 / Accepted 12 August 1994     

The action of exogenous gibberellic acid on isocitrate lyase —mRNA in germinating castor bean seeds

Gibberellic acid (GA₃) stimulates isocitrate lyase activity of the endosperm during germination of castor bean seeds. Isocitrate lyase from castor bean was purified and an antibody to it was prepared from rabbit serum. This antibody was used to measure the amounts of isocitrate lyase-mRNA using an in vitro translation system. No specific stimulation of isocitrate lyase-mRNA by application of GA₃ was detected. The stimulation of isocitrate lyase activity by exogenous GA₃ may be accounted for by the action of the growth substance in advancing the overall production of rRNA and mRNA which accelerates the rate of total protein synthesis during germination. The application of Amo 1618 retards the production of isocitrate lyase activity but also retards protein synthesis in general. This suggests that endogenous gibberellins also act non-specifically in the regulation of protein synthesis during castor bean germination.Abbreviations SDS sodium dodecyl sulphate - GA₃ gibberellic acid - PAGE polyacrylamide gel electrophoresis     

Spontaneous and reversible high-frequency frameshifts originating a phase transition in the carotenoid biosynthesis pathway of the phototrophic bacterium Rhodobacter sphaeroides 2.4.1

Summary The nucleotide sequence of the melB gene coding for the Na⁺(Li⁺)/melibiose symporter of Salmonella typhimurium LT2 was determined, and its amino acid sequence was deduced. It consists of 1428 bp, corresponding to a protein of 476 amino acid residues (calculated molecular weight 52800). The amino acid sequence is homologous to that of the melibiose permease of Escherichia coli K12, with 85% identical residues. All, except one, of the amino acid residues that have been reported to be important for cation or substrate recognition in the melibiose permease of E. coli are conserved in the melibiose permease of S. typhimurium. In addition, part of the sequence resembles the lactose permease of Streptococcus thermophilus, the animal glucose transporter (GLUT1), the plasmid-coded raffinose permease (RafB), and the NADH-ubiquinone oxidoreductase chain 4 (Nuo4) of Aspergillus amstelodami.The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank/EMBL Data Bank with accession number X62101     

Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.     

微小杆菌属(Exiguobacterium)细菌的能量代谢途径分析

【目的】微小杆菌属(Exiguobacterium)细菌广泛分布于海洋及非海洋环境中,具有多种代谢途径以适应复杂多样的生境。本研究从能量代谢途径角度出发,探究该属菌株对不同生境的适应能力。【方法】从美国国家生物科技数据中心(National Center for Biotechnology Information, NCBI)数据库中获取146个Exiguobacterium属菌株的基因组,查找并统计光营养、厌氧呼吸和底物代谢等多种能量代谢途径的关键蛋白或关键酶基因在各菌株基因组中的分布,包括光营养型的视紫红质基因、厌氧呼吸营养型的钼辅因子合成蛋白基因,以及底物代谢营养型中乙醛酸分流途径的异柠檬酸裂解酶及苹果酸合酶基因等。根据对应的氨基酸序列构建视紫红质、MoaC和异柠檬酸裂解酶的系统发育树,分析不同能量代谢途径在该属菌株进化过程中的保守性,推测其对于该属菌株的重要性。【结果】Exiguobacterium属中50%的种具有视紫红质基因,其中分离自非海洋生境的菌株更趋向于含有视紫红质基因。本研究所统计的全部非海洋生境菌株中,含有视紫红质基因的菌株占比约为70%,而在海洋生境菌株中该比例...     

A novel promoter,derived from the isocitrate lyase gene of Candida tropicalis,inducible with acetate in Saccharomyces cerevisiae

When the isocitrate lyase gene, containing 5'-upstream and 3'-flanking regions, of an n-alkane-assimilating yeast Candida tropicalis was introduced into Saccharomyces cerevisiae, the enzyme was functionally overexpressed in the cells grown on acetate. The amount of the recombinant isocitrate lyase expressed in S. cerevisiae was as much as 30% of the total soluble proteins in the cells, being comparable to that with GAL7 functional under the control of galactose. The expression was also observed when the cells were grown on glycerol, lactate, ethanol or oleate. These facts indicate that the isocitrate lyase gene upstream region (UPR-ICL) contains a strong promoter functional in S. cerevisiae. UPR-ICL is active as a promoter on cheap carbon sources such as acetate and nonconventional carbon sources such as oleate, whereas many conventional strong promoters demand relatively expensive sugars or sugars derivatives. Therefore, it is promising to construct an economical recombinant protein production system by using UPL-ICL.     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.     

Regulation of isocitrate lyase inRhodobacter capsulatus E1F1

InRhodobacter capsulatus E1F1, isocitrate lyase (ICL) (EC 4.5.3.1) is a regulatory enzyme whose levels are increased in the presence of acetate as the sole carbon source. Acetate activated isocitrate lyase in a process dependent on energy supply and de novo protein synthesis. In contrast to isocitrate lyase, isocitrate dehydrogenase (ICDH) activity was independent of the carbon source used for growth and significantly increased in darkened cells. Pyruvate or yeast extract prevented in vivo activation of isocitrate lyase in cells growing on acetate. The enzyme was reversibly inactivated to a great extent in vitro by pyruvate and other oxoacids presumably involved in acetate metabolism. These results suggest that, inR. capsulatus E1F1, isocitrate lyase is regulated by both enzyme synthesis and oxoacid inactivation.     

Itaconic acid: An inhibitor of isocitrate lyase in Tetrahymena pyriformis in vitro and in vivo

The succinate analog itaconic acid was observed to be a competitive inhibitor of the glyoxylate cycle specific enzyme isocitrate lyase (EC 4.1.3.1) in cell-free extracts of Tetrahymena pyriformis. Itaconic acid also inhibited net in vivo glycogen synthesis from glyoxylate cycle-dependent precursors such as acetate but not from glyoxylate cycle-independent precursors such as fructose. The effect of itaconic acid on the incorporation of ¹⁴C into glycogen from various ¹⁴C-labeled precursors was also consistent with inhibition of isocitrate lyase by this compound. Another analog of succinate which shares a common metabolic fate with itaconic acid, mesaconic acid, had no effect on isocitrate lyase activity in vitro or on ¹⁴C-labeled precursor incorporation into glycogen in vivo. In addition, itaconic acid did not affect gluconeogenesis from lactate in isolated perfused rat livers, a system lacking the enzyme isocitrate lyase. These results are taken as evidence that itaconic acid is an inhibitor of glyoxylate cycle-dependent glyconeogenesis Tetrahymena pyriformis via specific competitive inhibition of isocitrate lyase activity.     

Synthesis of Mono- and Sesquiterpenoids

The synthesis of isocitrate lyase in Candida tropicalis, the growth of which was stimulated by exogenously added biotin, was released from repression by glucose under biotin-deficient conditions. Biotin deficiency reduced remarkably the levels of biotin-enzymes, pyruvate carboxylase and acetyl-Co A carboxylase, in the glucose-utilizing cells of this yeast. A marked increase in intracellular level of pyruvate was observed in the biotin-deficient cells. Acetyl-CoA-donating compounds, such as pyruvate, acetate and alkanes, stimulated the formation of isocitrate lyase in the yeast regardless of the presence or absence of biotin. On the other hand, malate and succinate did not affect the enzyme synthesis. The isocitrate lyase synthesis under biotin-sufficient conditions was repressed by not only glucose but also glucosamine and 2-deoxyglucose. This repression by glucose was not eliminated by cAMP. The stimulated synthesis of isocitrate lyase under biotin-deficient conditions was also observed in C. albicans and C. guilliermondii growing on glucose.     

Gene cloning and transformation in the basidiomycete fungus Coprinus cinereus: Isolation and expression of the isocitrate lyase gene (acu-7)

Summary The cloned isocitrate lyase structural gene of Aspergillus nidulans (acuD) was shown to hybridize under reduced stringency conditions to unique sequences in genomic DNA digests of the basidiomycete fungus Coprinus cinereus. A gene library of C. cinereus was constructed in the lambda replacement vector L47 and screened for sequences hybridizing to the A. nidulans gene. A recombinant phage was isolated which contained the hybridizing sequence on a 5.6-kb BamHI fragment. This fragment was subcloned into pUC13 to give plasmid pHIONA1 and shown to contain a functional C. cinereus isocitrate lyase gene (acu-7) by transformation of an acu-7 mutant. Direct selection for Acu⁺ transformants was not possible because of the toxicity of the acetate selection medium. Acu⁺ transformants were obtained as cotransformants by transforming an acu-7 trp-1 double mutant, having mutations in both the isocitrate lyase and tryptophan synthetase structural genes, with a plasmid containing the trp-1 gene and either pHIONA1 or the original lambda clone. Up to 47.5% of the selected Trp⁺ transformants were cotransformed to Acu⁺. A physical analysis of 40 Acu⁺ transformants showed that the acu-7 gene had integrated at non-homologous and often multiple sites in the genome. Meiotic stability of the integrated gene was demonstrated by genetic crosses.     

The disappearance of isocitrate lyase from the green alga Chlorella fusca studied by immunoprecipitation

When acetate-adapted cultures of Chlorella fusca were transferred to nitrogen-free medium containing glucose, isocitrate lyase activity was lost over a period of about 25 h. Using a combination of in vivo isotope labelling and immunoprecipitation with anti-isocitrate lyase IgG it was shown that: 1. The onset of loss of enzyme activity preceeded the complete cessation of enzyme synthesis. 2. Disappearance of isocitrate lyase activity was accompanied by loss of enzyme protein, without accumulation of antigenic protein distinguishable from the normal subunit polypeptide of the enzyme, as judged by SDS gel electrophoresis of immunoprecipitated samples from supernatant cell-free extracts. 3. SDS gel electrophoresis of immunoprecipitated isocitrate lyase revealed the presence of antigenic protein bands of M_r about twice that of the normal subunit polypeptide, but the appearance of these apparent dimer forms did not obviously correlate with enzyme degradation. 4. Isoelectric focusing of immunoprecipitated isocitrate lyase showed that the enzyme became progressively more oxidised during the period of its degradation in vivo. 5. By titrating crude broken cell suspensions with anti-isocitrate lyase antibody, preliminary evidence was obtained for transfer of the enzyme from the soluble fraction to an insoluble form as part of the process of disappearance.     

Determination of genetic bases of auxotrophy in <Emphasis Type="Italic">Yersinia pestis</Emphasis> ssp. <Emphasis Type="Italic">caucasica</Emphasis> strains

Based on the results of computer analysis of nucleotide sequences in strains Yersinia pestis and Y. pseudotuberculosis recorded in the files of NCBI GenBank database, differences between genes argA, aroG aroF thiH, and thiG of strain Pestoides F (subspecies caucasica) were found, compared to other strains of plaque agent and pseudotuberculosis microbe. Using PCR with calculated primers and the method of sequence analysis, the structure of variable regions of these genes was studied in 96 natural Y. pestis and Y. pseudotuberculosis strains. It was shown that all examined strains of subspecies caucasica, unlike strains of plague-causing agent of other subspecies and pseudotubercolosis microbe, had identical mutations in genes argA (integration of the insertion sequence IS100), aroG (insertion of ten nucleotides), aroF (inserion of IS100), thiH (insertion of nucleotide T), and thiG (deletion of 13 nucleotides). These mutations are the reason for the absence in strains belonging to this subspecies of the ability to synthesize arginine, phenylalanine, tyrosine, and vitamin B1 (thiamine), and cause their auxotrophy for these growth factors.     

The Effects of Octanoate and Oleate on Isocitrate Lyase Activity during the Germination of Pinus pinea Seeds

The changes of isocitrate lyase levels with respect to the catabolism of triglycerides have been studied during the germination of Pinus pinea seeds. We studied the effects of octanoate, oleate, and inhibitors of protein synthesis on isocitrate lyase during germination. Pyruvate kinase, glucose-6-P-dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase were also assayed. Octanoate and oleate inhibited the isocitrate lyase activity, similarly to cycloheximide, chloramphenicol, and actinomycin, inhibitors of protein biosynthesis. This inhibitory effect is not specific but is strikingly evident with isocitrate lyase. This inhibition was not proportional to the concentration but was proportional to the chain length of oleate and octanoate.     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

Increased and decreased sensitivity to carbon catabolite repression of enzymes of acetate metabolism in mutants of Aspergillus nidulans.

Summary The creA204, creB15 and creC27 mutations have been shown to cause carbon catabolite derepression of acetyl CoA synthase and isocitrate lyase in Aspergillus nidulans. A recessive mutation, cre-34, which is linked to the creC gene, results in these enzymes being more sensitive than cre or wildtype strains to catabolite repression. The acetamidase levels of strains containing cre mutations have been investigated and provide support for the hypothesis that an acetate metabolite, rather than acetamide, induces this enzyme.