Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.     

Characterization of Sphingomonas paucimobilis SYK-6 genes involved in degradation of lignin-related compounds

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds. These compounds are degraded via vanillate and syringate by a unique enzymatic system, composed of etherases, O demethylases, ring cleavage oxygenases and side chain cleaving enzymes. These unique and specific lignin modification enzymes are thought to be powerful tools for utilization of the most abundant aromatic biomass, lignin. Here, we focus on the genes and enzymes involved in β-aryl ether cleavage and biphenyl degradation. Two unique etherases are involved in the reductive cleavage of β-aryl ether. These two etherases have amino acid sequence similarity with the glutathione S-transferases, and use glutathione as a hydrogen donor. It was found that 5,5′-dehydrodivanillate, which is a typical lignin-related biphenyl structure, was transformed into 5-carboxyvanillate by the reaction sequence of O-demethylation, meta-ring cleavage, and hydrolysis, and the genes involved in the latter two reactions have been characterized. Vanillate and syringate are the most common intermediate metabolites in lignin catabolism. These compounds are initially O-demethylated and the resulting diol compounds, protocatechuate (PCA) and 3-O-methylgallate, respectively, are subjected to ring cleavage catalyzed by PCA 4,5-dioxygenase. The ring cleavage products generated are further degraded through the PCA 4,5-cleavage pathway. We have isolated and characterized genes for enzymes involved in this pathway. Disruption of a gene for 2-pyrone-4,6-dicarboxylate hydrolase (ligI) in this pathway suggested that an alternative route for 3-O-methylgallate degradation, in which ligI is not involved, would play a role in syringate catabolism. In this article, we describe the genetic and biochemical features of the S. paucimobilis SYK-6 genes involved in degradation of lignin-related compounds. A possible application of the SYK-6 lignin degradation system to produce a valuable chemical material is also described. Received 01 May 1999/ Accepted in revised form 29 July 1999     

The Mycobacterium tuberculosis shikimate pathway genes: Evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases

Summary The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.     

Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z”

The genes involved in biosynthesis of the major compatible solute ectoine (1,4,5,6-tetrahydro-2-methylpyrimidine carboxylic acid) in halotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z” were studied. The complete nucleotide sequences of the structural genes encoding l-aspartokinase (Ask), l-2,4-diaminobutyric acid transaminase (EctB), l-2,4-diaminobutyric acid acetyltransferase (EctA), and l-ectoine synthase (EctC) were defined and shown to be transcribed as a single operon ectABCask. Phylogenetic analysis revealed high sequence identities (34–63%) of the Ect proteins to those from halophilic heterotrophs with the highest amino acid identities being to Vibrio cholerae enzymes. The chromosomal DNA fragment from “M. alcaliphilum 20Z” containing ectABC genes and putative promoter region was expressed in Escherichia coli. Recombinant cells could grow in the presence of 4% NaCl and synthesize ectoine. The data obtained suggested that despite the ectoine biosynthesis pathway being evolutionary well conserved with respect to the genes and enzymes involved, some differences in their organization and regulation could occur in various halophilic bacteria.Dedicated to the 70th birthday of Professor Gerhard Gottschalk who inspired our studies on methylotrophic haloalkaliphiles.     

Tryptophan catabolism via kynurenine production in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>: identification of three genes coding for the enzymes of tryptophan to anthranilate pathway

Most enzymes involved in tryptophan catabolism via kynurenine formation are highly conserved in Prokaryotes and Eukaryotes. In humans, alterations of this pathway have been related to different pathologies mainly involving the central nervous system. In Bacteria, tryptophan and some of its derivates are important antibiotic precursors. Tryptophan degradation via kynurenine formation involves two different pathways: the eukaryotic kynurenine pathway, also recently found in some bacteria, and the tryptophan-to-anthranilate pathway, which is widespread in microorganisms. The latter produces anthranilate using three enzymes also involved in the kynurenine pathway: tryptophan 2,3-dioxygenase (TDO), kynureninase (KYN), and kynurenine formamidase (KFA). In Streptomyces coelicolor, where it had not been demonstrated which genes code for these enzymes, tryptophan seems to be important for the calcium- dependent antibiotic (CDA) production. In this study, we describe three adjacent genes of S. coelicolor (SCO3644, SCO3645, and SCO3646), demonstrating their involvement in the tryptophan-to-anthranilate pathway: SCO3644 codes for a KFA, SCO3645 for a KYN and SCO3646 for a TDO. Therefore, these genes can be considered as homologous respectively to kynB, kynU, and kynA of other microorganisms and belong to a constitutive catabolic pathway in S. coelicolor, which expression increases during the stationary phase of a culture grown in the presence of tryptophan. Moreover, the S. coelicolor ΔkynU strain, in which SCO3645 gene is deleted, produces higher amounts of CDA compared to the wild-type strain. Overall, these results describe a pathway, which is used by S. coelicolor to catabolize tryptophan and that could be inactivated to increase antibiotic production.     

A gene cluster inChlorobium vibrioforme encoding the first enzymes of chlorophyll biosynthesis

A cloned 5.8-kb genomic fragment of the green sulfur bacteriumChlorobium vibrioforme encodes the genes for three enzymes catalyzing early steps in the biosynthetic pathway of tetrapyrroles, common to chlorophyll and heme. ThehemA, hemC andhemD genes encode the enzymes glutamyl tRNA dehydrogenase, porphobilinogen deaminase and uroporphyrinogen III synthase, respectively. The cloned genes were expressed in transformedEscherichia coli orSalmonella typhimurium and conferred autotrophy on the respective auxotrophs. Activities of the enzymes encoded by the cloned genes were demonstrated in vitro, with cell extracts obtained from the transformed enterobacteria. The proximity of these genes indicates that they form a cluster inChlorobium vibrioforme, while in most other organisms they appear to be scattered. The presence of this cluster may imply coordinate regulation of the genes involved and they may constitute an operon.     

Lactococcus lactis and stress

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis.Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase.To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37°C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.     

Characterization of the dTDP-rhamnose biosynthetic genes encoded in the rfb iocus of Shigella flexneri

The nucleotide sequence of the proximal half of the rfb region of Shigella flexneri has been determined, and the genes encoding enzymes involved in the biosynthesis of dTDP-rhamnose have been identified. These genes show strong homology to the rfb genes encoding dTDP-rhamnose biosynthesis in Salmonella enterica serovar typhimurium (strain LT2) and S. enterica serovar anatum (strain M32) (Jiang et al., 1991; Wang et al., 1992). An open reading frame upstream of rfbB was also identified which encoded a protein having strong similarity with GalU, and has been designated galF. GalF has 92% amino acid sequence identity with an S. enterica LT2 gene, orf2X8, which is similarly situated upstream of rfbB (Jiang et al., 1991). The T7 expression system was utilized to identify proteins corresponding to those predicted from DNA sequence analysis. The similarity of the predicted proteins with proteins that are functionally identical or related, and with others of unknown function from the Yersinia enterocolitica O3 rfb region, and in the Escherichia coli K-12 rff region are also described. We have re-addressed the assignment of each gene of the dTDP-rhamnose pathway with the known enzymes of the pathway, in particular rfbC and rfbD. A reporter plasmid to detect genes encoding enzymes of the dTDP-rhamnose pathway is described. An analysis of the intergenic region between galF and rfbB has been made, and comparison with the same region from S. enterica LT2 discussed.     

Molecular characterization and expression of 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose 5-phosphate reductoisomerase (DXR) gene from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes the first committed step of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plants. The present study describes the cloning and characterization of a cDNA encoding DXR from Salvia miltiorrhiza (designated as SmDXR, GenBank Accession No. FJ476255). Comparative and bioinformatic analyses revealed that SmDXR showed extensive homology with DXRs from other plant species. Phylogenetic tree analysis indicated that SmDXR belongs to the plant DXR superfamily and has the closest relationship with DXR from Lycopersicon esculentum. Tissue expression pattern analysis revealed that SmDXR expressed strongly in leaves, followed by roots and stems, implying that SmDXR was a constitutively expressed gene. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in tanshinone biosynthetic pathway in Salvia plants. The expression profiles revealed by RT-PCR under different elicitor treatments such as methyl jasmonate (MJ) and salicylic acid (SA) were compared for the first time, and the results revealed that SmDXR was an elicitor-responsive gene, which could be induced by SA in leaves and inhibited by exogenous MJ in three tested tissues. The functional color assay in Escherichia coli showed that SmDXR could accelerate the biosynthesis of lycopene, indicating that SmDXR encoded a functional protein. The characterization, expression profile and functional analysis of SmDXR gene will be helpful for further study in the role of SmDXR in tanshinones biosynthetic pathway and metabolic engineering to increase tanshinones production in S. miltiorrhiza.     

In vitro reconstitution of the catabolic reactions catalyzed by PcaHG,PcaB, and PcaL: the protocatechuate branch of the β-ketoadipate pathway in Rhodococcus jostii RHA1

The β-ketoadipate pathway is a major pathway involved in the catabolism of the aromatic compounds in microbes. The recent progress in genome sequencing has led to a rapid accumulation of genes from the β-ketoadipate pathway in the available genetic database, yet the functions of these genes remain uncharacterized. In this study, the protocatechuate branch of the β-ketoadipate pathway of Rhodococcus jostii was reconstituted in vitro. Analysis of the reaction products of PcaHG, PcaB, and PcaL was achieved by high-performance liquid chromatography. These reaction products, β-ketoadipate enol-lactone, 3-carboxy-cis,cis-muconate, γ-carboxymuconolactone, muconolactone, and β-ketoadipate, were further characterized using LC-MS and nuclear magnetic resonance. In addition, the in vitro reaction of PcaL, a bidomain protein consisting of γ-carboxy-muconolactone decarboxylase and β-ketoadipate enol-lactone hydrolase activities, was demonstrated for the first time. This work provides a basis for analyzing the catalytic properties of enzymes involved in the growing number of β-ketoadipate pathways deposited in the genetic database.     

A tobacco cDNA clone encoding a GATA-1 zinc finger protein homologous to regulators of nitrogen metabolism in fungi

In higher plants, the expression of the nitrate assimilation pathway is highly regulated. Although the molecular mechanisms involved in this regulation are currently being elucidated, very little is known about the trans-acting factors that allow expression of the nitrate and nitrite reductase genes which code for the first enzymes in the pathway. In the fungus Neurospora crassa, nit-2, the major nitrogen regulatory gene, activates the expression of unlinked structural genes that specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein containing a single zinc finger motif defined by the C-X₂-CX₁₇-C-X₂-C sequence. This DNA-binding domain recognizes the promoter region of N. crassa nitrogen-related genes and fragments derived from the tomato nia gene promoter. The observed specificity of the binding suggests the existence of a NIT2-like homolog in higher plants. PCR and cross-hybridization techniques were used to isolate, respectively, a partial cDNA from Nicotiana plumbaginifolia and a full-length cDNA from Nicotiana tabacum. These clones encode a NIT2-like protein (named NTL1 for nit-2-like), characterized by a single zinc finger domain, defined by the C-X₂-C-X₁₈-C-X₂-C amino acids, and associated with a basic region. The amino acid sequence of NTL1 is 60% homologous to the NIT2 sequence in the zinc finger domain. The Ntl1 gene is present as a unique copy in the diploid N. plumbaginifolia species. The characteristics of Ntl1 gene expression are compatible with those of a regulator of the nitrate assimilation pathway, namely weak nitrate inducibility and regulation by light.     

碱性条件下苏云金芽胞杆菌基础代谢分析

【目的】探索苏云金芽胞杆菌(Bacillus thuringiensis)形成转录差异的碱性条件,明确B.thuringiensis在该条件下的基础代谢途径变化。【方法】采用半定量RT-PCR技术及实时荧光定量PCR技术,确定碱刺激下参考基因psp A存在表达差异的碱性处理条件。在该条件下提取RNA进行Agilent定制B.thuringiensis表达谱芯片杂交,对芯片数据进行差异表达分析、GO富集分析及生物途径富集分析等。【结果】通过检测psp A表达变化,将对数生长中期的菌体加入终浓度为28 mmol/L的Na OH并诱导培养10min,作为B.thuringiensis响应碱刺激的研究条件。富集分析表明碳代谢、脂肪酸合成代谢、氨基酸合成代谢途径变化明显。细胞糖酵解途径至少19个酶促基因上调表达,三羧酸循环中催化α-酮戊二酸转化为苹果酸的大部分酶蛋白编码基因上调2倍以上。【结论】本研究发现在碱性条件下B.thuringiensis基础代谢明显增强,细胞可能通过大量合成酸性物质如乳酸、苹果酸等来提高细胞对于碱性环境的适应能力。     

Improved production of adipate with Escherichia coli by reversal of β-oxidation

The linear C₆ dicarboxylic acid adipic acid is an important bulk chemical in the petrochemical industry as precursor of the polymer nylon-6,6-polyamide. In recent years, efforts were made towards the biotechnological production of adipate from renewable carbon sources using microbial cells. One strategy is to produce adipate via a reversed β-oxidation pathway. Hitherto, the adipate titers were very low due to limiting enzyme activities for this pathway. In most cases, the CoA intermediates are non-natural substrates for the tested enzymes and were therefore barely converted. We here tested heterologous enzymes in Escherichia coli to overcome these limitations and to improve the production of adipate via a reverse β-oxidation pathway. We tested in vitro selected enzymes for the efficient reduction of the enoyl-CoA and in the final reaction for the thioester cleavage. The genes encoding the enzymes which showed in vitro the highest activity were then used to construct an expression plasmid for a synthetic adipate pathway. Expression of paaJ, paaH, paaF, dcaA, and tesB in E. coli BL21(DE3) resulted in the production of up to 36 mg/L of adipate after 30 h of cultivation. Beside the activities of the pathway enzymes, the availability of metabolic precursors may limit the synthesis of adipate, providing another key target for further strain engineering towards high-yield production of adipate with E. coli.