Horizontally Acquired Cellulases Assist the Expansion of Dietary Range in Pristionchus Nematodes.

Horizontal gene transfer (HGT) enables the acquisition of novel traits via non-Mendelian inheritance of genetic material. HGT plays a prominent role in the evolution of prokaryotes, whereas in animals, HGT is rare and its functional significance is often uncertain. Here, we investigate horizontally acquired cellulase genes in the free-living nematode model organism Pristionchus pacificus. We show that these cellulase genes 1) are likely of eukaryotic origin, 2) are expressed, 3) have protein products that are secreted and functional, and 4) result in endo-cellulase activity. Using CRISPR/Cas9, we generated an octuple cellulase mutant, which lacks all eight cellulase genes and cellulase activity altogether. Nonetheless, this cellulase-null mutant is viable and therefore allows a detailed analysis of a gene family that was horizontally acquired. We show that the octuple cellulase mutant has associated fitness costs with reduced fecundity and slower developmental speed. Furthermore, by using various Escherichia coli K-12 strains as a model for cellulosic biofilms, we demonstrate that cellulases facilitate the procurement of nutrients from bacterial biofilms. Together, our analysis of cellulases in Pristionchus provides comprehensive evidence from biochemistry, genetics, and phylogeny, which supports the integration of horizontally acquired genes into the complex life history strategy of this soil nematode.

Effects of catechin polyphenols and preparations from the plant-parasitic nematode Heterodera glycines on protease activity and behaviour in three nematode species.

Protease activities in preparations from the plant-parasitic nematodes Heterodera glycines and Meloidogyne incognita and the free-living nematode Panagrellus redivivus were inhibited by exposure to a series of eight catechin polyphenol analogues, (+)-catechin, (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-gallocatechin gallate (GCG), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) (1 mm each), and by a preparation from H. glycines cysts. General protease activity detected with the FRET-peptide substrate QXL520-KSAYMRF-K(5-FAM)a and proteasome chymotrypsin-like (CTL) activity detected with succinyl-LLVY-AMC were each inhibited significantly more (P < 0.05) by the gallated form of the polyphenol than by the corresponding non-gallated form. Species differences in response to inhibition across all analogues were revealed with the CTL substrate, but CG was a consistently potent inhibitor across all three species and with each substrate. A heat-stable component (CE) from H. glycines cysts inhibited M. incognita CTL activity by 92.07 ± 0.68%, significantly less (P < 0.05) in H. glycines (52.86 ± 2.77%), and by only 17.24 ± 0.55% (P < 0.05) in P. redivivus preparations. CTL activity was, however, inhibited more than 60% in all preparations by the proteasome-specific inhibitor MG-132. Hatching of M. incognita infective juveniles exposed to 1 mm CG, ECG, GCG or EGCG was reduced by 83.88 ± 4.26%, 69.98 ± 9.14%, 94.93 ± 1.71% and 87.93 ± 2.89%, respectively, while hatching of H. glycines was reduced less than 25% by each analogue. CE had no effect on nematode hatch, but did cause a 60% reduction in mobility of H. glycines infective juveniles exposed overnight to CE in vitro, which was more (P < 0.05) than the reduction of M. incognita infective juvenile mobility (20%).

Cloning and molecular analysis of the aspartic protease Sc-ASP110 gene transcript in Steinernema carpocapsae.

Many protease genes have previously been shown to be involved in parasitism and in the development of Steinernema carpocapsae, including a gene predicted to encode an aspartic protease, Sc-ASP110, which was cloned and was analysed in this study. A cDNA encoding Sc-ASP110 was cloned based on an expressed sequence tag (EST) fragment from our EST library. The full-length cDNA of Sc-ASP110 consists of 1112 nucleotides with a catalytic aspartic domain (aa18-337). The putative 341 amino acid residues have a calculated molecular mass of 37·1 kDa and a theoretical pI of 4·7. BLASTp analysis of the Sc-ASP110 amino acid sequence showed 45-77% amino acid sequence identity to parasitic and non-parasitic nematode aspartic proteases. An expression analysis showed that the sc-asp110 gene was upregulated during the late parasitic stage, L4, and 24 h after induction of in vitro nematodes. A sequence comparison revealed that Sc-ASP110 was a member of an aspartic protease family; additionally, a phylogenetic analysis indicated that Sc-ASP110 was clustered with the closely related nematode Steinernema feltiae. In situ hybridization showed that sc-asp110 was expressed in the body walls of dorsal cells. The upregulated Sc-ASP110 expression revealed that this protease could play a role in the late parasitic process. In this study, we have cloned and analysed the gene transcript of Sc-ASP110 in S. carpocapsae.

In vitro proteolysis of nematode FMRFamide-like peptides (FLPs) by preparations from a free-living nematode (Panagrellus redivivus) and two plant-parasitic nematodes (Heterodera glycines and Meloidogyne incognita).

Proteolytic activities in extracts from three nematodes, the plant parasites Heterodera glycines and Meloidogyne incognita, and the free-living Panagrellus redivivus, were surveyed for substrate preferences using a battery of seven FRET-modified peptide substrates, all derived from members of the large FMRF-amide like peptide (FLP) family in nematodes. Overall protease activity in P. redivivus was four- to fivefold greater than in either of the parasites, a result that might reflect developmental differences. Digestion of the M. incognita FLP KHEFVRFa (substrate Abz-KHEFVRF-Y(3-NO2)a) by M. incognita extract was sevenfold greater than with H. glycines extract and twofold greater than P. redivivus, suggesting species-specific preferences. Additional species differences were revealed upon screening 12 different protease inhibitors. Two substrates were used in the screen, Abz-KHEFVRF-Y(3-NO2)a and Abz-KPSFVRF-Y(3-NO2)a), which was digested equally by all three species. The effects of various inhibitor, substrate and extract source combinations on substrate digestion suggest that M. incognita differs significantly from P. redivivus and H. glycines in its complement of cysteine proteases, particularly cathepsin L-type protease.

Biochemical study and in vitro insect immune suppression by a trypsin-like secreted protease from the nematode Steinernema carpocapsae.

A trypsin-like serine protease was purified by gel filtration and anion-exchange chromatography from the excretory-secretory products of parasitic phase Steinernema carpocapsae. The purified protease exhibited a molecular mass of about 29 kDa by SDS-PAGE and displayed a pI of 6.3. This protease exhibited high activity with trypsin-specific substrate N-Ben-Phe-Val-Arg-p-nitroanilide and was highly sensitive to aprotinin and benzamidine. The purified trypsin protease digested the chromogenic substrate N-Ben-Phe-Val-Arg-p-nitroanilide with K(m), V(max) and k(cat) values of 594.2 mum, 0.496 mum/min and 22.8/s, respectively. The optimal pH and temperature for protease activity were 9 and 30 degrees C, respectively. Internal amino acid sequencing yielded 150 amino acids and these were homologous to other trypsin sequences. In vitro investigation was carried out to monitor prophenoloxidase suppression in Galleria mellonella by the purified protease; about 38.9-52.6% suppression of prophenoloxidase was observed. The purified protease affected insect haemocyte spreading, causing cells to become spherical or round. Protease-treated actin filaments were highly disorganized in haemocytes. In vitro, G. mellonella haemocytes recognized infective juveniles of Heterorhabditis bacteriophora; however, S. carpocapsae and Steinernema glaseri were not recognized. We provide experimental evidence that the purified trypsin has the potential to alter host haemocytes, actin filaments and to inhibit host haemolymph melanization.

In vitro comparison of protease activities in preparations from free-living (Panagrellus redivivus) and plant-parasitic (Meloidogyne incognita) nematodes using FMRFa and FMRFa-like peptides as substrates.

Extracts prepared from the microbivorous nematode Panagrellus redivivus and the plant-parasitic nematode Meloidogyne incognita were used to provide general protease activities for peptide substrate screening and species comparisons. Each extract was evaluated for its ability to degrade a broad range of nematode FMRFamide-like peptides (FLPs), key regulatory messengers governing nematode growth and development. Clear quantitative differences between the two extracts were observed using FMRFamide as a substrate. Extract potency assessed at EC50 (µg/µ l extract protein for 50% substrate digestion) was 1.8-fold greater for P. redivivus than for M. incognita, and potency assessed at EC90 was 2.5-fold greater. An overall potency difference was also present when screening the digestion of 17 nematode FLPs, but it was not universal. The mean percentage digestion of eight of the 17 FLPs was greater (P < 0.02) with P. redivivus extract (76.3 ± 8.2) than with M. incognita extract (38.1 ± 8.7), but the means for the other nine FLPs were not different. Three FLPs (KPSFVRFa, AQTFVRFa, RNKFEFIRFa) were degraded extensively by the extracts of both species, and two FLPs (SAPYDPNFLRFa, SAEPFGTMRFa) were degraded 2.9-fold and 5.3-fold greater, respectively, with M. incognita extract than with P. redivivus extract. The ability of each extract to degrade FMRFa and KSAYMRFa was significantly reduced by using peptide analogues containing single d-amino acid substitutions, and the substitution effects were positional. Both FMRFa and KSAYMRFa were competitive substrates for aminopeptidases in each extract, but only the competitive ability of FMRFa was reduced by d-amino acid substitution. The variety and complexity of nematode FLP degradation by preparations representing phylogenetically and developmentally different nematode sources are discussed.

Identification and expression analysis of the Steinernema carpocapsae elastase-like serine protease gene during the parasitic stage.

A cDNA encoding elastase was isolated from Steinernema carpocapsae by suppression subtractive hybridization and rapid amplification of 5' cDNA ends. The predicted protein contained a 19-aa signal peptide, a 44-aa N-terminal propeptide, and a 264-aa mature protein with a predicted molecular mass of 28,949 Da and a theoretical pI of 8.88. BLAST analysis showed 27-35% amino acid sequence identity to serine proteases from insects, mammals, fish and other organisms. The Sc-ela gene contains three exons and two introns with at least two copies in the S. carpocapsae genome. Expression analysis indicated that the Sc-ela gene was upregulated during the initial parasitic stage. Sequence comparison and evolutionary marker analysis revealed that Sc-ELA was a member of the elastase serine protease family with potential degradative, developmental and fibrinolytic activities. Homology modeling showed that Sc-ELA adopts a two beta-barrel fold typical of trypsin-like serine proteases, and phylogenetic analysis indicates that Sc-ELA branched off early during elastase evolution.

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp.

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30min, nematodes were observed adhered after 40min and many were captured by the typical fungus traps after 70min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.

Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans.

Similar to other plant-parasitic nematodes, root lesion nematodes possess an array of enzymes that are involved in the degradation of the plant cell wall. Here we report the identification of a gene encoding a cell wall-degrading enzyme, pectin methylesterase PME (EC 3.1.1.11), in the root lesion nematode Pratylenchus penetrans. Both genomic and coding sequences of the gene were cloned for this species, that included the presence of four introns which eliminated a possible contamination from bacteria. Expression of the Pp-pme gene was localized in the esophageal glands of P. penetrans as determined by in situ hybridization. Temporal expression of Pp-pme in planta was validated at early time points of infection. The possible function and activity of the gene were assessed by transient expression of Pp-pme in plants of Nicotiana benthamiana plants via a Potato virus X-based vector. To our knowledge, this is the first report on identification and characterization of a PME gene within the phylum Nematoda.

Molecular identification and functional characterization of the cathepsin B gene (Ab-cb-1) in the plant parasitic nematode Aphelenchoides besseyi.

The rice white tip nematode, Aphelenchoides besseyi, is widely distributed in rice planting areas worldwide and causes serious economic losses. Cathepsin genes have been demonstrated to have importance in studying the reproduction, development, pathogenicity, and control methods of plant nematodes. In this paper, a novel cathepsin B gene, Ab-cb-1, was found and cloned. The Ab-cb-1 gene was 1347 bp in length and encodes 369 amino acids. The Ab-CB-1 protein contains characteristic occluding loops but no signal peptide. A homology analysis showed that Ab-CB-1 had the highest identity value (64%) to the known amino acid sequence of cathepsin B-like cysteine protease 6 from Toxocara canis. When Ab-cb-1 was expressed in a prokaryotic system, the protein massed approximately 45 kDa and could decompose carrot callus. Ab-cb-1 mRNA was localized in the nematode intestine. The relative expression level of Ab-cb-1 in the A. besseyi Ab-S24 population, which had high reproductivity, was approximately 6.9 times that in the Ab-N10 population, which had low reproductivity, and the difference was significant (p<0.05). The Ab-cb-1 expression level was highest in females; the expression levels in males, juveniles and eggs were 30%, 12.2% and 5% of that in females, respectively, and the differences were significant among all four stages (p<0.05). Nematodes of the Ab-S24 population were treated with Ab-cb-1 dsRNA for 12 h, 24 h, 36 h and 48 h, and their reproduction decreased with increasing time. These results demonstrated that Ab-CB-1 was a digestive enzyme with hydrolytic protease properties and that Ab-cb-1 played an important role in the reproduction of A. besseyi.

A sulfotransferase dosage-dependently regulates mouthpart polyphenism in the nematode Pristionchus pacificus.

Polyphenism, the extreme form of developmental plasticity, is the ability of a genotype to produce discrete morphologies matched to alternative environments. Because polyphenism is likely to be under switch-like molecular control, a comparative genetic approach could reveal the molecular targets of plasticity evolution. Here we report that the lineage-specific sulfotransferase SEUD-1, which responds to environmental cues, dosage-dependently regulates polyphenism of mouthparts in the nematode Pristionchus pacificus. SEUD-1 is expressed in cells producing dimorphic morphologies, thereby integrating an intercellular signalling mechanism at its ultimate target. Additionally, multiple alterations of seud-1 support it as a potential target for plasticity evolution. First, a recent duplication of seud-1 in a sister species reveals a direct correlation between genomic dosage and polyphenism threshold. Second, inbreeding to produce divergent polyphenism thresholds resulted in changes in transcriptional dosage of seud-1. Our study thus offers a genetic explanation for how plastic responses evolve.

Protein interaction of nucleosome assembly protein 1 and casein kinase 2 during desiccation response in the insect-killing nematode Steinernema feltiae IS-6.

The change in gene expression induced by desiccation in the semiarid, entomopathogenic nematode Steinernema feltiae IS-6, includes induction of transcription of a nucleosome assembly protein, NAP1 homolog, and of casein kinase 2 (CK2) genes. Therefore, one of the events during the dehydration response of S. feltiae IS-6 may be transcriptional activation by S. feltiae IS-6 NAP1 homolog (Sf-Nap1), which is regulated by S. feltiae IS-6 CK2 (Sf-CK2). This regulation necessitates physical interaction between the Sf-Nap1 and Sf-CK2 proteins. In the present study we used yeast 2-hybrid analysis to demonstrate physical interaction between the 2 proteins, thus confirming the involvement of a protein interaction-based step in the desiccation response mechanism of S. feltiae IS-6.

Alterations in the levels of glycogen and glycogen synthase transcripts during desiccation in the insect-killing nematode Steinernema feltiae IS-6.

The ability to withstand desiccation by entering anhydrobiosis is important for the survival of many nematode species. We are interested in the metabolic changes that occur during dehydration in the semiarid strain IS-6 of the insect parasitic nematode Steinernema feltiae. These changes may enable IS6 to be more tolerant to desiccation than temperate strains. We identified genes of IS-6 that exhibit changes in transcript levels during dehydration. These included glycogen synthase (Sf-gsy-1), which is the rate-limiting enzyme in the synthesis of glycogen, which is likely to play a role in desiccation survival. We established the changes in the steady state level of Sf-gsy-1 transcripts upon dehydration and determined the biochemical changes in the level of its product, glycogen, during the dehydration and rehydration of nematodes. Our results suggest a shift from glycogen to trehalose synthesis during dehydration, which is regulated at least in part by suppression of glycogen synthase transcription.

Steinernema glaseri surface enolase: molecular cloning, biological characterization, and role in host immune suppression.

Entomopathogenic nematodes are widely used as biological control agents that can suppress or evade the host immune defense upon entry into insects. The surface coat of Steinernema glaseri has been shown to play important roles in defeating the host immune system. In this work, a protein fraction with antiphagocytic activity was separated by electro-elution and further analyzed by two-dimensional electrophoresis (2-DE). LC-MS/MS analysis of one protein spot from a 2-DE gel gave five peptides that were highly similar to enolases of many organisms. A 1311 bp cDNA was cloned that encodes a 47 kDa protein with high sequence identity to enolases from different species of nematodes. The deduced protein, Sg-ENOL, was expressed in Escherichia coli, and its glycolytic activity was demonstrated by the conversion of 2-phospho-d-glycerate to phosphoenolpyruvate. Recombinant Sg-ENOL significantly reduced the LT(50)s of Xenorhabdus poinarii and Metarhizium anisopliae when co-injected into Galleria mellonella and Locusta migratoria manilensis Meyen, respectively. Using immuno-gold transmission electron microscopy, native Sg-ENOL was confirmed to be localized to both the nematode cuticle and the surface coat. In vitro, secretion of Sg-ENOL was inducible rather than constitutive. In vivo, Sg-ENOL was detected in the host hemolymph after infection of G. mellonella with S. glaseri, indicating that Sg-ENOL was secreted into the insect hemocoel and was involved in infection. This is the first report of the cloning and characterization of a surface coat protein in an entomopathogenic nematode. Our findings provide clear evidence for an important role for a cell surface enolase in S. glaseri infection and host immune suppression.

Pepsin-like aspartic protease (Sc-ASP155) cloning, molecular characterization and gene expression analysis in developmental stages of nematode Steinernema carpocapsae.

Steinernema carpocapsae is an insect parasitic nematode associated with the bacterium Xenorhabdus nematophila. These symbiotic complexes are virulent against the insect host. Many protease genes were shown previously to be induced during parasitism, including one predicted to encode an aspartic protease, which was cloned and analyzed in this study. A cDNA encoding Sc-ASP155 was cloned based on the EST fragment. The full-length cDNA of Sc-ASP155 consists of 955 nucleotides with multiple domains, including a signal peptide (aa1-15), a pro-peptide region (aa16-45), and a typical catalytic aspartic domain (aa71-230). The putative 230 amino acid residues have a calculated molecular mass of 23,812Da and a theoretical pI of 5.01. Sc-ASP155 blastp analysis showed 40-62% amino acid sequence identity to aspartic proteases from parasitic and free-living nematodes. Expression analysis showed that the sc-asp155 gene was up-regulated during the initial parasitic stage, especially in L3 gut and 6h induced nematodes. Sequence comparison revealed that Sc-ASP155 was a member of an aspartic protease family and phylogenetic analysis indicated that Sc-ASP155 was clustered with Sc-ASP113. In situ hybridization showed that sc-asp155 was expressed in subventral cells. Additionally, we determined that sc-asp155 is a single-copy gene in S. carpocapsae. Homology modeling showed that Sc-ASP155 adopts a typical aspartic protease structure. The up-regulated Sc-ASP155 expression revealed that this protease could play a role in the parasitic process. In this study, we have cloned the gene and determined the expression of the pepsin-like aspartic protease Sc-ASP155 in S. carpocapsae.

Cloning, characterisation and heterologous expression of an astacin metalloprotease, Sc-AST, from the entomoparasitic nematode Steinernema carpocapsae.

Steinernema carpocapsae is a parasitic nematode that is high virulent to insects. The parasitic juvenile reaches the insect haemocoelium by passing through mid-gut barriers and develops there. During invasion, the nematode was predicted to express a large set of proteases, including metalloproteases, one of which was sequenced and expressed in this work. A 1583-nucleotide cDNA encoding a putative metalloprotease containing a 28-aa signal peptide, a 79-aa propeptide and a 311-aa mature protease with a predicted molecular mass of 35.2 kDa and a theoretical pI of 5.9 was cloned from the parasitic stage of the nematode. Sequence analyses predicted signature sequences of the astacin metalloprotease family, an astacin domain, a zinc-binding motif and a methionine turn motif; therefore, this protein was identified as an astacin and designated as Sc-AST. The astacin domain of Sc-AST has an amino acid sequence homology of 46% to prototypical astacin from Astacus astacus and 82% to Caenorhabditis elegans NAS-8. Like NAS-8 of C. elegans, Sc-AST has a C-terminal ShK toxin domain. Recombinant Sc-AST was produced in an Escherichia coli system and was purified by affinity chromatography. Maldi-MS/MS analysis of purified recombinant protein matched the Sc-AST sequence with a significance score of 499. Sc-AST was produced in the correct folding conformation, showed activities against gelatin and azocasein substrates and was inhibited by divalent metal-chelating agents. Sc-AST presented an optimum pH of 7.5 and temperature of 37°C and K(m), V(max) and k(cat) values of 1.86 mM, 0.281 µM/min and 27.9 s(-1), respectively. Expression analyses indicated that Sc-AST is up-regulated in the parasitic stage and is strongly induced in vitro by insect tissues, thus suggesting that it plays a role in the parasitic process.

An apoptosis-inducing serine protease secreted by the entomopathogenic nematode Steinernema carpocapsae.

Steinernema carpocapsae is an insect parasitic nematode able to parasitise and kill the host within 48 h. Secreted products (ESP) of the parasitic stage of a virulent strain contain higher amounts of proteolytic activity than a low virulence strain, suggesting proteases are involved in virulence. From the ESP we purified a protein (Sc-SP-3) with a M(r) of 30 kDa and a pI of 7 that cleaved the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA and was inhibited by phenylmethanesulfonyl fluoride, benzamidine and chymostatin, thus indicating that it belongs to the chymotrypsin-like serine protease family. Sc-SP-3 has a V(max) of 0.3 mM min(-1)ml(-1) and K(m) of 6.6 x 10(-4)M, with maximum activity at pH 8 and 40 degrees C. The full-length cDNA was obtained using degenerate oligonucleotides for serine proteases. This open reading frame encodes a preproprotein containing a putative signal peptide composed of 16 amino acid residues, a prodomain of 40 residues and a mature protease domain of 261 residues, including the catalytic triad His/Asp/Ser characteristic of trypsin-like serine proteases. The N-terminal sequence and the peptide masses fingerprint obtained by MALDI-TOF-MS for the purified protein matched the cDNA. Gene expression analysis by quantitative real-time-PCR showed that this gene is expressed only during the parasitic stage and that pre-invasive nematodes inside the mid-gut expressed higher amounts of Sc-SP-3 than those that already enter the haemocoel. Sc-SP-3 caused histolysis in the insect mid-gut. In vitro assays demonstrated that Sc-SP-3 digested extracellular proteins and induced apoptosis in Sf9 insect cells, thus suggesting Sc-SP-3 is a multifunctional chymotrypsin-like protease involved in pathogenesis.

Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein.

A southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp. In the 5' non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified. The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni(2+)-chelating affinity chromatography. The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa. On the basis of gel filtration analyses it is shown that the active enzyme is a dimer. The specific enzyme activity was determined to be 4.2 mumol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity. The Km value for L-ornithine was determined as 44 microM. The Ki values for putrescine, alpha-diffluoromethylornithine, alpha-hydrazino-ornithine and alpha-methylornithine were calculated as 51, 34, 0.34 and 42 microM respectively.