Azotobacter vinelandii gene clusters for two types of peptidic and catechol siderophores produced in response to molybdenum.

AIM: To characterize the complementary production of two types of siderophores in Azotobacter vinelandii. METHODS AND RESULTS: In an iron-insufficient environment, nitrogen-fixing A. vinelandii produces peptidic (azotobactin) and catechol siderophores for iron uptake to be used as a nitrogenase cofactor. Molybdenum, another nitrogenase cofactor, was also found to affect the production level of siderophores. Wild-type cells excreted azotobactin into molybdenum-supplemented and iron-insufficient medium, although catechol siderophores predominate in molybdenum-free environments. Two gene clusters were identified to be involved in the production of azotobactin and catechol siderophores through gene annotation and disruption. Azotobactin-deficient mutant cells produced catechol siderophores under the molybdenum-supplemented and iron-insufficient conditions, whereas catechol siderophore-deficient mutant cells extracellularly secreted excess azotobactin under iron-deficient condition independent of the concentration of molybdenum. This evidence suggests that a complementary siderophore production system exists in A. vinelandii. CONCLUSIONS: Molybdenum was found to regulate the production level of two types of siderophores. Azotobacter vinelandii cells are equipped with a complementary production system for nitrogen fixation in response to a limited quantity of metals. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study identifying A. vinelandii gene clusters for the biosynthesis of two types of siderophores and clarifying the relationship between them.

Transport of dopamine and levodopa and their interaction in COS-7 cells heterologously expressing monoamine neurotransmitter transporters and in monoaminergic cell lines PC12 and SK-N-SH.

The present study investigated the effects of levodopa, a precursor of dopamine (DA) therapeutically used for the treatment of Parkinson's disease, on DA transport in the two different systems, COS-7 cells heterologously expressing rat monoamine transporter cDNA and in monoaminergic cell lines PC12 and SK-N-SH. Levodopa enhanced uptake of [3H]DA and [3H]norepinephrine (NE) but not [3H]serotonin in the transfected COS-7 cells in a concentration-dependent manner. On the other hand, in PC12 and SK-N-SH cells where NET is functionally expressed, levodopa enhanced [3H]DA and [3H]NE uptake at low concentrations and inhibited the uptake at higher concentrations. The effects of levodopa on catecholamine transporters in the opposite direction suggest a different mechanism at the intra- and extracellular sites in a levodopa transport-dependent and independent manner.

Therapeutic and specific antitumor immunity induced by co-administration of immature dendritic cells and adenoviral vector expressing biologically active IL-18.

Interleukin-18 is a potent cytokine expressed early in the immune response following cleavage in activated composes. We have investigated the in vivo antitumor effects of intratumoral (i.t.) administration of an adenoviral vector expressing biologically active murine interleukin (IL)-18 (Ad.PTH.IL-18). Substantial antitumor effects were observed when established MCA205 fibrosarcoma was treated in syngeneic immunocompetent mice with intratumoral injection of Ad.PTH.IL-18 (P = 0.0025 versus control vector treatment), generating potent cytotoxic T lymphocytes (CTLs) in culture. In contrast, the antitumor effect was absent, and cytotoxic activity was significantly less (P = 0.021) in gld mice (Fas ligand deficient). To enhance the in vivo antitumor activity of the treatment using Ad.PTH.IL-18, we co-injected immature DC and Ad.PTH.IL-18 i.t. into established, day 7 MCA205 fibrosarcoma and MC38 adenocarcinoma. Co-injection of both Ad.PTH.IL-18 and DC was associated with complete abrogation of injected tumors. Furthermore, the antitumor effects were also observed on distant tumors inoculated i.d. in the contralateral flank of the animal. The induced cytolytic activity was tumor-specific and MHC class I-restricted. As we have previously demonstrated in vitro (Tanaka F et al, Cancer Res 2000; 60: 4838-4844) and consistent with these findings in vivo, NK, T and dendritic cells coactivately mediate the IL-18 enhanced antitumor effect. This study suggests that the coactivate strategy could be used in the clinical setting to treat patients with cancer. do

Super-channel in bacteria: function and structure of the macromolecule import system mediated by a pit-dependent ABC transporter.

In a soil isolate, Sphingomonas sp. A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter. The transporter is different from other ABC transporters so far analyzed in that its function is dependent on a pit, a mouth-like organ formed on the cell surface only when cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells. The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated the 'super-channel', and in this review, we discuss the three-dimensional structure and specific function of the 'super-channel' for macromolecule import found for the first time in a bacterium.

Super-channel in bacteria: function and structure of a macromolecule import system mediated by a pit-dependent ABC transporter.

In a soil isolate, Sphingomonas sp. A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter. The transporter is different from other ABC transporters so far analyzed in that its function is dependent on the pit, a mouth-like organ formed on the cell surface only when the cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells. The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated as the "Super-channel", and in this review, we discuss the three-dimensional structure and specific function of the "Super-channel" for macromolecule import found for the first time in a bacterium.

Molecular characterization of Escherichia coli NAD kinase.

NAD kinase was purified to homogeneity from Escherichia coli MG1655. The enzyme was a hexamer consisting of 30 kDa subunits and utilized ATP or other nucleoside triphosphates as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 7.5 and 60 degrees C. The enzyme could not use inorganic polyphosphates as phosphoryl donors and was designated as ATP-NAD kinase. The N-terminal amino-acid sequence of the purified enzyme was encoded by yfjB, which had been deposited as a gene of unknown function in the E. coli whole genomic DNA sequence database. yfjB was cloned and expressed in E. coli BL21(DE3)pLysS. The purified product (YfjB) showed NAD kinase activity, and was identical to ATP-NAD kinase purified from E. coli MG1655 in molecular structure and other enzymatic properties. The deduced amino-acid sequence of YfjB exhibited homology with that of Mycobacterium tuberculosis inorganic polyphosphate/ATP-NAD kinase [Kawai, S., Mori, S., Mukai, T., Suzuki, S., Hashimoto, W., Takeshi, Y. & Murata, K. (2000) Biochem. Biophys. Res. Commun. 276, 57-63], and those of many hypothetical proteins for which functions have not yet been revealed. The YfjB homologues were considered to be NAD kinases and alignment of their sequences revealed highly conserved regions, XXX-XGGDG-XL and DGXXX-TPTGSTAY, where X represents a hydrophobic amino-acid residue.

Crystallization and preliminary X-ray analysis of AlgS, a bacterial ATP-binding cassette (ABC) protein specific to macromolecule import.

Sphingomonas sp. A1 possesses a macromolecule (alginate; average molecular size 25 700 Da) uptake system mediated by a novel pit-dependent ABC transporter. In this system, AlgS (363 amino-acid residues; 40 kDa) functions as an ATPase and provides energy for the translocation of high molecular-weight alginate across the cytoplasmic membrane. Hexahistidine-tagged AlgS of Sphingomonas sp. A1 was overexpressed in Escherichia coli and crystallized by means of the hanging-drop vapour-diffusion method with ammonium dihydrogen monophosphate as the precipitant. Preliminary X-ray analysis of the resultant crystals was performed; they belonged to the monoclinic space group P2(1) and had unit-cell parameters a = 57.4, b = 92.7, c = 65.8 A, beta = 102.3 degrees. X-ray diffraction data to 3.2 A have been collected from the native crystal.

Crystal structure of alginate lyase A1-III complexed with trisaccharide product at 2.0 A resolution.

The structure of A1-III from a Sphingomonas species A1 complexed with a trisaccharide product (4-deoxy-l-erythro-hex-4-enepyranosyluronate-mannuronate-mannuronic acid) was determined by X-ray crystallography at 2.0 A with an R-factor of 0.16. The final model of the complex form comprising 351 amino acid residues, 245 water molecules, one sulfate ion and one trisaccharide product exhibited a C(alpha) r.m.s.d. value of 0.154 A with the reported apo form of the enzyme. The trisaccharide was bound in the active cleft at subsites -3 approximately -1 from the non-reducing end by forming several hydrogen bonds and van der Waals interactions with protein atoms. The catalytic residue was estimated to be Tyr246, which existed between subsites -1 and +1 based on a mannuronic acid model oriented at subsite +1.

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1.

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.

Crystal structure of N-acyl-D-glucosamine 2-epimerase from porcine kidney at 2.0 A resolution.

The X-ray crystallographic structure of N-acyl-d-glucosamine 2-epimerase (AGE) from porcine kidney, which has been identified to be a renin-binding protein (RnBP), was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 16.9 % for 15 to 2.0 A resolution data. The refined structure of AGE comprised 804 amino acid residues (one dimer) and 145 water molecules. The dimer of AGE had an asymmetric unit with approximate dimensions 46 Ax48 Ax96 A. The AGE monomer is composed of an alpha(6)/alpha(6)-barrel, the structure of which is found in glucoamylase and cellulase. One side of the AGE alpha(6)/alpha(6)-barrel structure comprises long loops containing five short beta-sheets, and contributes to the formation of a deep cleft shaped like a funnel. The putative active-site pocket and a possible binding site for the substrate N-acetyl-d-glucosamine (GlcNAc) were found in the cleft. The other side of the alpha(6)/alpha(6)-barrel comprises short loops and contributes to the dimer formation. At the dimer interface, which is composed of the short loops and alpha-helices of the subunits, five strong ion-pair interactions were observed, which play a major role in the dimer assembly. This completely ruled out the previously accepted hypothesis that the formation of the RnBP homodimer and RnBP-renin heterodimer requires the leucine zipper motif present in RnBP.

Safety assessment of rice genetically modified with soybean glycinin by feeding studies on rats.

Feeding studies on rice genetically modified with soybean glycinin were performed on rats for four weeks. The rats were divided into three groups, each being fed on (I) only a commercial diet, (II) this diet plus control rice and (III) this diet plus rice genetically modified with glycinin. The rats were fed with 10 g/kg-weight of rice every day by oral administration. During the test period, the rats in every group grew well without marked differences in appearance, food intake, body weight, or cumulative body weight gain. There were also no significant differences in the blood count, blood composition or internal organ weights among the rats. Necropsy at the end of the experiment indicated neither pathological symptoms nor histopathological abnormalities in the liver and kidney. Judging from these results, the rice genetically modified with glycinin is considered to have been essentially the same in nutritional and biochemical characteristics as the control rice.

Inorganic Polyphosphate/ATP-NAD kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv.

An enzyme with both inorganic polyphosphate [poly(P)]- and ATP-dependent NAD kinase activities was isolated from Micrococcus flavus. The enzyme was a dimer consisting of 34 kDa subunits, and was named poly(P)/ATP-NAD kinase. Internal amino acid sequences of the enzyme showed homologies with some function-unknown proteins released on the GenBank database. Among such proteins, hypothetical Rv1695 protein (Accession No. Z98268-16), which was encoded by a gene named "Rv1695" on genomic DNA of Mycobacterium tuberculosis H37Rv, was proposed to be poly(P)-dependent NAD kinase. By cloning and expression in Escherichia coli, Rv1695 was shown to encode poly(P)/ATP-NAD kinase and named ppnk. The ppnk product, recombinant-poly(P)/ATP-NAD kinase (Ppnk) was purified and characterized. The enzyme was a tetramaer consisting of 35 kDa subunits when expressed in E. coli. Poly(P)/ATP-NAD kinases of M. flavus and Ppnk of M. tuberculosis H37Rv specifically and completely phosphorylated NAD by utilizing commercially available poly(P)s and nucleoside triphosphates as phosphoryl donors.

Rapid generation of potent and tumor-specific cytotoxic T lymphocytes by interleukin 18 using dendritic cells and natural killer cells.

We hypothesized that antitumor-specific immunity, which is induced by interleukin (IL) 18 treatment in murine tumor models, is promoted by enhancing natural killer (NK)-mediated destruction of tumor and delivery to dendritic cells (DCs). These activated and antigen-pulsed DCs then critically and optimally induce an adoptive immune response, positioning IL-18 as an important bridge between the innate and adoptive immune response. The effect of IL-18 added to cultures of live tumor cells (MCA205, a mouse sarcoma cell line), NK cells, DCs, and T cells was assessed. When recombinant (r) mIL-18 protein was added to this culture, potent NK cytolytic activity with subsequent generation of CTLs was observed in a dose-dependent manner. Without introduction of either rmIL-18 or NK cells into this culture, systemic cytolytic activity was significantly decreased. Following the absence of direct contact of either NK cells or DCs with other cells in this cooperative coculture system using transwell, the systemic cytolytic activity of both NK cells and CTLs was greatly suppressed. The cytolysis mediated by effector cells harvested after completion of the culture was primarily restricted to MHC class I and highly specific for the tumor cells used in the coculture. Furthermore, we examined the efficiency in the induction of cytolytic T cells of other established IFN-gamma inducing T-cell growth factors, IL-2, and IL-12 in this culture system and compared them with that mediated by IL-18. Neither IL-2 nor IL-12 induced tumor-specific cytolytic T cells to the same degree as that mediated by IL-18. Efficacy of this system in induction of tumor-specific CTLs was also observed in the system using MC38 adenocarcinoma cells. These results are consistent with the notion that IL-18 induces tumor-specific immunity through enhancing NK activity, which in turn mediates tumor cell death and activates and primes DCs.

Properties of basis functions generated by shift invariant sparse representations of natural images.

The idea that a sparse representation is the computational principle of visual systems has been supported by Olshausen and Field [Nature (1996) 381: 607-609] and many other studies. On the other hand neurons in the inferotemporal cortex respond to moderately complex features called icon alphabets, and such neurons respond invariantly to the stimulus position. To incorporate this property into sparse representation, an algorithm is proposed that trains basis functions using sparse representations with shift invariance. Shift invariance means that basis functions are allowed to move on image data and that coefficients are equipped with shift invariance. The algorithm is applied to natural images. It is ascertained that moderately complex graphical features emerge that are not as simple as Gabor filters and not as complex as real objects. Shift invariance and moderately complex features correspond to the property of icon alphabets. The results show that there is another connection between visual information processing and sparse representations.

Molecular identification of oligoalginate lyase of Sphingomonas sp. strain A1 as one of the enzymes required for complete depolymerization of alginate.

A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37 degrees C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (aly) and the ABC transporter (algS, algM1, and algM2). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.

A novel bacterial ATP-binding cassette transporter system that allows uptake of macromolecules.

A gram-negative bacterium, Sphingomonas sp. strain A1, isolated as a producer of alginate lyase, has a characteristic cell envelope structure and forms a mouth-like pit on its surface. The pit is produced only when the cells have to incorporate and assimilate alginate. An alginate uptake-deficient mutant was derived from cells of strain A1. One open reading frame, algS (1,089 bp), exhibiting homology to the bacterial ATP-binding domain of an ABC transporter, was cloned as a fragment complementing the mutation. algS was followed by two open reading frames, algM1 (972 bp) and algM2 (879 bp), which exhibit homology with the transmembrane permeases of ABC transporters. Disruption of algS of strain A1 resulted in the failure to incorporate alginate and to form a pit. Hexahistidine-tagged AlgS protein (AlgS(His6)) overexpressed in Escherichia coli and purified by Ni(2+) affinity column chromatography showed ATPase activity. Based on these results, we propose the occurrence of a novel pit-dependent ABC transporter system that allows the uptake of macromolecules.

Overexpression in Escherichia coli, purification, and characterization of Sphingomonas sp. A1 alginate lyases.

A bacterium Sphingomonas sp. A1 produces three kinds of alginate lyases [A1-I (66 kDa), A1-II (25 kDa), and A1-III (40 kDa)] from a single precursor, through posttranslational processing. Overexpression systems for these alginate lyases were constructed in Escherichia coli cells by controlling of the lyase genes under T7 promoter and terminator. Expression levels of A1-I, A1-II, and A1-III in E. coli cells were 3.50, 3.04, and 2.13 kU/liter of culture, respectively, and were over 10-fold higher than those in Sphingomonas sp. A1 cells. Purified A1-I, A1-II, and A1-III from E. coli cells were monomeric enzymes with molecular masses of 63, 25, and 40 kDa, respectively. The depolymerization pattern of alginate with A1-I and A1-II indicated that both enzymes cleaved the glycosidic bond of the polymer endolytically and by beta-elimination reaction. A1-II preferred polyguluronate rather than polymannuronate and released tri- and tetrasaccharides, which have unsaturated uronyl residues at the nonreducing terminal, from alginate as the major final products. A1-I acted equally on both homopolymers and produced di- and trisaccharides as the final products.

Crystallization and preliminary X-ray crystallographic analysis of alginate lyase A1-II from Sphingomonas species A1.

Alginate lyase A1-II of Sphingomonas species A1 was purified and crystallized using the hanging drop vapor-diffusion method in 0.1 M Tris-HCl buffer containing 43% saturated ammonium sulfate, 8% polyethylene glycol 4000 and 0.2 M Li(2)SO(4) at pH 8.5 and 20 degrees C. The crystals are tetragonal and belong to the space group P4(3)2(1)2 or P4(1)2(1)2 with unit cell dimensions of a=b=144.07 and c=296.38 A. The diffraction data up to 2.8 A were collected by a synchrotron radiation source at SPring-8 in Japan.

Potent antitumor effects mediated by local expression of the mature form of the interferon-gamma inducing factor, interleukin-18 (IL-18).

IL-18 is produced during the acute immune response by macrophages and immature dendritic cells. IL-18 receptors are induced on T cells and NK cells by IL-12 and together they enhance a cellular immune response. We constructed retroviral and adenoviral vectors encoding the mature bioactive murine IL-18 in order to examine their immune and antitumor effects in murine tumor models. Secretion of bioactive IL-18 from murine tumor cells was facilitated by transfecting them with recombinant viral vectors carrying the prepro leader sequence of human parathyroid hormone fused to the 5' end of the mature murine IL-18 cDNA. Direct injection of the IL-18 recombinant adenoviral vector (Ad.PTH.IL-18) into an established MCA205 murine fibrosarcoma completely eradicated tumor in all animals with concomitant induction of protective systemic immunity. Co-administration of systemic IL-12 provided synergistic antitumor effects when combined with peritumoral injections of Ad.PTH.IL-18 without apparent side-effects as we observed with systemic administration of IL-18. Depletion of asialo GM-1+ cells completely abrogated the antitumor effects of Ad.PTH.IL-18, suggesting a major role for NK cells in mediating the anti-tumor effects of IL-18. Peritumoral injection of Ad.PTH.IL-18 was also associated with reduced numbers of CD8+ cells found within the tumor (HBSS versus Ad.PTH.IL-18, P < 0.0001). This suggests that IL-18 could be utilized as an alternative cancer gene therapy especially when combined with systemic administration of IL-12.

Unsaturated glucuronyl hydrolase of Bacillus sp. GL1: novel enzyme prerequisite for metabolism of unsaturated oligosaccharides produced by polysaccharide lyases.

The bacterium Bacillus sp. GL1 assimilates two kinds of heteropolysaccharides, gellan and xanthan, by using extracellular gellan and xanthan lyases, respectively, and produces unsaturated saccharides as the first degradation products. A novel unsaturated glucuronyl hydrolase (glycuronidase), which was induced in the bacterial cells grown on either gellan or xanthan, was found to act on the tetrasaccharide of unsaturated glucuronyl-glucosyl-rhamnosyl-glucose produced from gellan by gellan lyase, and the enzyme and its gene were isolated from gellan-grown cells. The nucleotide sequence showed that the gene contained an ORF consisting of 1131 base pairs coding a polypeptide with a molecular weight of 42,859. The purified enzyme was a monomer with a molecular mass of 42 kDa and was most active at pH 6.0 and 45 degrees C. Because the enzyme can act not only on the gellan-degrading product by gellan lyase, but also on unsaturated chondroitin and hyaluronate disaccharides produced by chondroitin and hyaluronate lyases, respectively, it is considered that the unsaturated glucuronyl hydrolase plays specific and ubiquitous roles in the degradation of oligosaccharides with unsaturated uronic acid at the nonreducing terminal produced by polysaccharide lyases.