Molecular Cloning of the leuB Gene from Bacteroides fragilis by Functional Complementation in Escherichia coli

Clones containing the Bacteroides fragilis leuB-complementing gene were isolated by screening of a B. fragilis genomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5 kb) could complement three independent leuB mutations in E. coli and the leuB-complementing determinant in pOT865 was localized to a region of 1.5-kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilis genome. The cloned fragment appeared to contain a sequence that could function as a promoter in E. coli and direct the synthesis of a 42-kDa protein. These results suggest that the cloned segment contains the structural gene for β-isopropylmalate dehydrogenase (leuB).     

Cloning and Characterization of the NAD-Dependent 7α-Hydroxysteroid Dehydrogenase from <Emphasis Type="Italic">Bacteroides fragilis</Emphasis>

The NAD-linked 7-hydroxysteroid dehydrogenase (7-HSDH) from Bacteroides fragilis ATCC 25285 was characterized and its gene cloned. The enzyme displayed optimal activities at pH 8.5 (NAD reduction) and 6.5 (NADH oxidation). The lowest K_m and highest V_max values were observed with chenodeoxycholic acid and its conjugates. The protein had subunits of 27.4 kDa and a native size of 110 kDa, suggesting a homotetrameric composition. The enzyme was relatively thermostable, retaining 95% of initial activity after 1 h at 65°C. A DNA probe based on the N-terminal amino acid sequence hybridized to a 2373-bp HindIII fragment of B. fragilis DNA. This fragment was cloned into E. coli and sequenced, revealing a 780-bp open reading frame. The predicted amino acid sequence of the ORF showed strong sequence similarity to three other bacterial 7-HSDHs, all in the short-chain dehydrogenase family. The regulation of expression of this gene is currently under investigation.     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

Isolation of a gene encoding 3-isopropylmalate dehydrogenase from rice

3-Isopropylmalate dehydrogenase (IPMDH, EC 1.1.1.85) is a key enzyme in the biosynthesis of leucine and glucosinolates in plants. IPMDH is a bifunctional dimeric enzyme that catalyzes dehydrogenation and decarboxylation reactions in the presence of NAD⁺. The leuB gene encoding IPMDH has been identified in a variety of bacteria and some plants. In this study, we analyze the gene for IPMDH from Oryza sativa (OsIPMDH). The analysis of an EST sequence and rice genome revealed a full-length open reading frame encoding 389 amino acids that correspond to a protein of approximately 41.2 kD. The predicted amino acid sequence of OsIPMDH was highly homologous with the IPMDHs from plants and bacteria. The OsIPMDH expression analysis in a leuB mutant of Escherichia coli revealed that OsIPMDH was capable of functionally complementing the leuB mutant. These results indicate that OsIPMDH encodes for a protein of 3-isopropylmalate dehydrogenase in rice.     

Characterization of the Cα-Dehydrogenase Gene Involved in the Cleavage of β-Aryl Ether by Pseudomonas paucimobilis

Cα-dehydrogenase catalyzes the oxidation of arylglycerol-β-aryl ether at the Ca-position, and therefore this process produces the specific substrate for β- etherase, which cleaves β-ary ethers (Ca carbonyl type).

Here we isolated the Ca-dehydrogenase gene (ligD) and sequenced its nucleotides. This gene contains an open reading frame of 915 bp and the deduced amino acid sequence had a homology with the ribitol dehydrogenase family. ligD is about 1 kbp upstream of the β-etherase gene (ligE).     

Identification of the Metallo-β-Lactamase Gene from Clinical Isolates of Bacteroides fragilis

Bacteroides fragilis is one of the organisms known to produce carbapenem-hydrolysing metallo-β-lactamase, which can confer resistance to a wide variety of β-lactams. The purpose of this study was to identify carbapenem-hydrolysing metallo-β-lactamase-producing B. fragilis strains by means of PCR assay, nucleotide sequencing and enzyme inhibition studies. Ten β-lactam-resistant B. fragilis isolates were investigated. Four imipenem-resistant strains among the 10 isolates gave positive reactions in the PCR assay. The nucleotide sequences of the PCR products from two imipenem-resistant strains shared >98% similarity with the metallo-β-lactamase gene from B. fragilis TAL 3636, which was used as a control. The amino acid sequence homology between the two imipenem-resistant strains and B. fragilis TAL 3636 was 99.2%. These strains produced high amounts of Zn²⁺-dependent β-lactamases which were inactivated by EDTA.     

Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase

Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.     

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

Restriction mapping and partial characterization of β-isopropylmalate dehydrogenase gene cloned from Citrobacter freundii

The β-isopropylmalate dehydrogenase gene of Citrobacter freundii was cloned in both Escherichia coli and Bacillus subtilis using pLS353 as a vector and PstI as an enzyme. The molecular weight of pCBL 1–7 containing β-isopropylmalate dehydrogenase gene was 3.4–7.7 megadalton (Md) and the restriction enzyme patterns of the plasmids were analyzed with enzymes such as BaII, BgII, EcoRI and SstII. The enzyme activities in both E. coli and B. subtilis transformants were 4–8-fold higher than those in the present strains.     

Characterization of two β-decarboxylating dehydrogenases from <Emphasis Type="Italic">Sulfolobus acidocaldarius</Emphasis>

Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg²⁺, thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.     

Sequence Analysis of the Plasmid pRRI2 from the Rumen Bacterium Prevotella ruminicola 223/M2/7 and the Use of pRRI2 in Prevotella/Bacteroides Shuttle Vectors

pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4.     

Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution.

We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus. The resulting transformant showed a leucine-autotrophy at 56 degrees C but not at 61 degrees C and above. Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme.     

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus

A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP⁺. Optimal temperature for activity was 90° C or above, and a half-life of 22 min was found for the enzyme when incubated at 90° C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups. Received: 6 February 1997 / Accepted: 12 June 1997     

Complete amino acid sequence of Bombyx egg-specific protein deduced from cDNA clone

Complementary (c)DNA coding for an insect yolk protein, the egg-specific protein of the silkworm Bombyx mori was cloned and the nucleotide sequence determined. The sequence covers the entire coding region of 1,677 base pairs with 5′ and 3′ noncoding regions (21 and 115 base pairs, respectively). The deduced amino acid sequence of the egg-specific protein consists of 559 amino acid residues. The NH₂-terminal 18 amino acid sequence is enriched in hydrophobic amino acids and assumed to be a signal peptide. A sequence, Asn-X-Thr, a potential N-linked glycosylation site, is found at positions 191 to 193. A serine-rich domain is localized in the region from 63 to 90, in which phosphorylation takes place. Cys His motif in 405 to 415 is analogous to a proposed metal binding sequence. Lys¹³²-Asn¹³³ and Arg²²⁸-Asp²²⁹ are probably the sites cleaved by the egg-specific protein protease that appears during embryogenesis. The derived amino acid sequence has no appreciable homology to other sequenced proteins.     

Molecular cloning and nucleotide sequence of 3-isopropylmalate dehydrogenase gene (leuB) from an extreme thermophile, Thermus aquaticus YT-1

A gene (leuB) coding for 3-isopropylmalate dehydrogenase [EC 1.1.1.85] from an extreme thermophile, Thermus aquaticus YT-1 was cloned in Escherichia coli and the nucleotide sequence was determined. It contains an open reading frame of 1,035 bp encoding 344 amino acid residues. The homology with that from T. thermophilus HB8 is 87.0% in nucleotide and 91.3% in amino acid sequences. No overlapped gene was found in the present leuB gene, in contrast to the previous prediction that Thermus leuD gene is overlapped with leuB [Croft et al. (1987) Mol. Gen. Genet. 210, 490-497]. Substitutions in the primary structure which are unique for the thermophile sequences are discussed in relation to the unusual stability of the thermophile dehydrogenase based on amino acid sequence comparison of 9 microorganisms including thermophiles and mesophiles.     

Screening of stable proteins in an extreme thermophile, Thermus thermophilus

The leuB gene codes for 3-isopropylmalate dehydrogenase of the leucine biosynthetic pathway in an extreme thermophile, Thermus thermophilus. The leuB gene of the thermophile was replaced with a temperature-sensitive chimeric leuB gene. The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained. A single base substitution that replaces isoleucine at 93 with leucine was found in the chimeric leuB gene of the thermostable mutant. The resultant amino acid residue coincided with the corresponding residue of the T. thermophilus enzyme. It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme. This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.     

Cloning and Characterization of the Gene Encoding Pyrroloquinoline Quinone-dependent Poly (vinyl alcohol) Dehydrogenase of Pseudomonas sp. Strain VM15C

A gene library of poly (vinyl alcohol) (PVA)-degrading Pseudomonas sp. strain VM15C was constructed in Escherichia coli with the vector pUC18. Screening of this library with a chromogenic PVA dehydrogenase assay resulted in the isolation of a clone that carries the gene (pdh) for the PVA dehydrogenase, and the entire nucleotide sequence of its structural gene was determined. The gene encodes a protein of 639 amino acid residues (68,045 Da) and in the deduced amino acid sequence, some putative functional sites, a signal sequence, a heme c-binding site, and a PQQ-binding site, were detected. The amino acid sequence showed low similarity to other types of quinoprotein dehydrogenases. PVA dehydrogenase expressed in E. coli clones required PQQ. Ca²⁺, and Mg²⁺ stimulated the activity. PVA-dependent heme c reduction occurred with exogenous PQQ in cell extracts of the E. coli clone. The PVA dehydrogenase in the E. coli clone was localized in the cytoplasm.     

Comprehensive metabolite profiling of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> using gas chromatography-mass spectrometry

A metabolite analysis of the soil bacterium Sinorhizobium meliloti was established as a first step towards a better understanding of the symbiosis with its host plant Medicago truncatula. A crucial step was the development of fast harvesting and extraction methods for the bacterial metabolites because of rapid changes in their composition. S. meliloti 1021 cell cultures grown in minimal medium were harvested by centrifugation, filtration or immediate freezing in liquid nitrogen followed by a lyophilisation step. Bacteria were lysed mechanically in methanol and hydrophilic compounds were analysed after methoxymation and silylisation via GC-MS. The different compounds were identified by comparison with the NIST 98 database and available standards. From about 200 peaks in each chromatogram 65 compounds have been identified so far. A comparison of the different extraction methods giving the metabolite composition revealed clear changes in several amino acids and amino acid precursor pools. A principal component analysis (PCA) was able to distinguish S. meliloti cells grown on different carbon sources based on their metabolite profile. A comparison of the metabolite composition of a S. meliloti leucine auxotrophic mutant with the wild type revealed a marked accumulation of 2-isopropylmalate in the mutant. Interestingly, the accumulated metabolite is not the direct substrate of the mutated enzyme, 3-isopropylmalate dehydrogenase, but the substrate of isopropylmalate isomerase, which acts one step further upstream in the biosynthetic pathway of leucine. This finding further emphasises the importance of integrating metabolic data into post-genomic research.