Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Quantitative Structure-Activity Relationships for the Pre-Steady State of <Emphasis Type="Italic">Pseudomonas Species</Emphasis> Lipase Inhibitions by <Emphasis Type="Italic">p</Emphasis>-Nirophenyl-<Emphasis Type="Italic">N</Emphasis>-Substituted Carbamates

The pre-steady states of Pseudomonas species lipase inhibitions by p-nitrophenyl-N-substituted carbamates (1–6) are composed of two steps: (1) formation of the non-covalent enzyme–inhibitor complex (E:I) from the inhibitor and the enzyme and (2) formation of the tetrahedral enzyme–inhibitor adduct (E–I) from the E:I complex. From a stopped-flow apparatus, the dissociation constant for the E:I complex, K_S, and the rate constant for formation of the tetrahedral E–I adduct from the E:I complex, k₂ are obtained from the non-linear least-squares of curve fittings of first-order rate constant (k_obs) versus inhibition concentration ([I]) plot against k_obs=k₂+k₂[I]/(K_S+[I]). Values of pK_S, and log k₂ are linearly correlated with the σ^* values with the ρ^* values of −2.0 and 0.36, respectively. Therefore, the E:I complexes are more positive charges than the inhibitors due to the ρ^* value of −2.0. The tetrahedral E–I adducts on the other hand are more negative charges than the E:I complexes due to the ρ^* value of 0.36. Formation of the E:I complex from the inhibitor and the enzyme are further divided into two steps: (1) the pre-equilibrium protonation of the inhibitor and (2) formation of the E:I complex from the protonated inhibitor and the enzyme.     

Phenyl methyl ethers: novel electron donors for respiratory growth of<Emphasis Type="Italic"> Desulfitobacterium hafniense</Emphasis> and<Emphasis Type="Italic"> Desulfitobacterium</Emphasis> sp. strain PCE-S

Desulfitobacterium hafniense and Desulfitobacterium sp. strain PCE-S grew under anoxic conditions with a variety of phenyl methyl ethers as electron donors in combination with fumarate as electron acceptor. The phenyl methyl ethers were O-demethylated to the corresponding phenol compounds. O-demethylation was strictly dependent on the presence of fumarate; no O-demethylation occurred with CO₂ as electron acceptor. One mol phenyl methyl ether R-O-CH₃ was O-demethylated to R-OH per 3 mol fumarate reduced to succinate. The growth yields with vanillate or syringate plus fumarate were approximately 15 g cells (dry weight) per mol methyl moiety converted. D. hafniense utilized vanillate or syringate as an electron donor for reductive dehalogenation of 3-Cl-4-hydroxyphenylacetate, whereas strain PCE-S was not able to dechlorinate tetrachloroethene with phenyl methyl ethers. Crude extracts of both organisms showed O-demethylase activity in the O-demethylase assay with vanillate or syringate as substrates when the organism was grown on syringate plus fumarate. Besides the homoacetogenic bacteria, only growing cells of Desulfitobacterium frappieri PCP-1 have thus far been reported to be capable of phenyl methyl ether O-demethylation. This present study is the first report of Desulfitobacteria utilizing phenyl methyl ethers as electron donors for fumarate reduction and for growth.Abbreviations PCE Tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - ClOHPA 3-Cl-4-Hydroxyphenylacetate - OHPA 4-Hydroxyphenylacetate - FH₄ Tetrahydrofolate     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.     

Characterization of osmolyte betaine synthesizing sarcosine dimethylglycine <Emphasis Type="Italic">N</Emphasis>-methyltransferase from <Emphasis Type="Italic">Methanohalophilus portucalensis</Emphasis>

To overcome the extracellular salt stress, Methanohalophilus portucalensis FDF1^T synthesizes the compatible solute betaine through the methylation of glycine, sarcosine, and N,N-dimethylglycine. S-adenosylmethionine (AdoMet) is the methyl donor. The enzyme sarcosine dimethylglycine N-methyltransferase (SDMT) of M. portucalensis, that catalyzes the formation of N,N-dimethylglycine and glycine betaine, has been purified and characterized. SDMT, a monomer of 33 kDa with a pI at 5.03, has a narrow substrate specificity limited to using only sarcosine and dimethylglycine as substrates for the methyl transferase reaction. The K _m values for sarcosine and AdoMet were 2.29 and 0.21 mM, respectively, with a V _max of 0.83 μmol/mg-min (k _cat value of 0.44 s⁻¹). The K _m values for dimethylglycine and AdoMet were 3.76 and 0.59 mM, respectively, with a V _max of 4.88 μmol/mg-min (k _cat of 2.68 s⁻¹). A high concentration of the end product betaine (2.0 M) did not affect the SMT activity, but it slightly inhibited the DMT activity. Both activities were also not affected by potassium or sodium ions in concentrations of 200–1,000 mM. We compared this novel archaeal SDMT enzyme to other similar bacterial transferases as well as to the glycine sarcosine dimethylglycine methyltransferase found also in M. portucalensis.     

The <Emphasis Type="Italic">b</Emphasis><Subscript>arg36</Subscript> contributes to efficient coupling in F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli, the F₁F_O ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the b _arg36→ile and the b _arg36→glu mutations. The b _arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b _arg36→glu mutation was largely suppressed in the b _{arg36→glu,glu39→arg} double mutant. E. coli expressing the b _{arg36→glu,glu39→arg} subunit grew well on succinate-based medium. F₁F_O ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F₁F_O ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Coenzyme precursor-assisted expression of a cholesterol oxidase from <Emphasis Type="Italic">Brevibacterium</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene (choB _b ), encoding cholesterol oxidase from Brevibacterium sp. CCTCC M201008, was cloned and sequenced by PCR (GenBank accession number: DQ345780). The gene consists of 1653 base pairs and encodes a protein of 551 amino acids. ChoB _b exhibited a homology of 98% with cholesterol oxidase gene from Brevibacterium sterolicum ATCC 21387. The cholesterol oxidase gene, cloned in the vector pET-28a, was over-expressed in Escherichia coli BL21–CodonPlus (DE3)-RP grown at 23°C in Luria-Bertani medium containing 50 μM riboflavin, the precursor of the FAD coenzyme of the enzyme. A maximum activity of 3.7 U/mg was obtained from cell free extract of E. coli BL21-CodonPlus (DE3)-RP harboring the pET-28a-choB_b.     

Functional characterization of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> with alterations in the <Emphasis Type="Italic">atpE</Emphasis> gene

The FUD17 strain of Chlamydomonas reinhardtii is a photosynthesis-deficient, acetate-requiring mutant with a defect in the chloroplast atpE gene, which codes for the ε subunit of the chloroplast ATP synthase. In this work, the FUD17 mutant was examined in relation to other known ATP synthase mutants as an initial step toward using this strain to generate altered versions of the atpE gene for site-directed mutagenesis of the ε subunit. The FUD17 strain grows well and is normally pigmented in the dark (heterotrophic conditions), but cannot grow autotrophically in the light, even when media are supplemented with acetate. Under heterotrophic conditions, it shows no accumulation of the ε subunit, and much lower levels of the α and β subunits of the chloroplast ATP synthase. FUD17 shows no light-dependent oxygen evolution and shows a strong, light-dependent alteration in its chlorophyll fluorescence. These results show that FUD17 possesses similar characteristics to other ATP synthase mutants and fails to express an assembled ATP synthase complex on its thylakoid membrane. A preliminary attempt at site-directed mutagenesis is described which produced a slightly truncated form of the ε subunit, which is expressed normally in the cell. This revised version was published online in June 2006 with corrections to the Cover Date.     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Removal of <Emphasis Type="Italic">p</Emphasis>-chlorophenol by the marine microalga <Emphasis Type="Italic">Tetraselmis marina</Emphasis>

The ability of Tetraselmis marina, a green coastal microalga, to remove chlorophenols under photoautotrophic conditions was investigated. T.marina was able to grow in the presence of 20 mg L⁻¹ of the phenolic compounds tested. The EC₅₀ (growth rate) value of p-chlorophenol (p-CP) to T.marina was found to be 25.5 mg L⁻¹. The microalga was able to remove chlorophenols, showing higher efficiency for p-CP. The effect of photoregime and NaHCO₃ concentration on p-CP removal was investigated. Under continuous illumination with 1 g L⁻¹ NaHCO₃ initial concentration T.marina removed 65% of 20 mg L⁻¹ in a 10-day cultivation period.     

Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

<Emphasis Type="Italic">n</Emphasis>-Alkanes variability in the diazotrophic cyanobacterium <Emphasis Type="Italic">Anabaena cylindrica</Emphasis> in response to NaCl stress

n-Alkanes pattern in response to NaCl stress has been studied in the cyanobacterium Anabaena cylindrica. Saturated hydrocarbons were separated and identified by gas chromatography-mass spectrometry (GC-MS) using serially coupled capillary column. Light chain n-alkanes in the range of C₉–C₁₇ (43%) and heavy chain n-alkanes in range of C₁₇–C₂₃ (34%) and C₂₃–C₃₁ (23%) were identified as the major components of total hydrocarbons in the NaCl adapted cells of A. cylindrica. In contrast, NaCl-untreated cells of A. cylindrica had dominance of only long chain n-alkanes in the range of C₂₃–C₃₁ comprising about 94% of its total n-alkanes. The persistence of high level (43%) of short chain n-alkanes (C₉–C₁₇) in NaCl adapted cells of A. cylindrica as compared to its negligible level (0.2%) in NaCl untreated counterpart clearly indicates that NaCl stress causes the A. cylindrica to shift towards the synthesis of short chain n-alkanes.     

Optimization of <Emphasis Type="Italic">Renealmia mexicana</Emphasis> (Klotzsch ex. Petersen) cultivation <Emphasis Type="Italic">in vitro</Emphasis>

Renealmia mexicana (Klotzsch ex. Petersen) is a tropical plant found in southern México with an ornamental value and a potential source of curcuminoids. Its distribution in Chiapas has decreased because of deforestation and low propagation and germination rate, so a protocol for in vitro propagation was developed. An orthogonal experimental design of L₉ (3⁴) in triplicate was used to investigate the effect of 6-benzyl adenine (BA), indole butyric acid (IBA), silver nitrate (AgNO₃), and sucrose on shoot, root, and leaf development of plantlets grown in vitro. Plantlets with well-developed shoots and roots were transferred to pots containing a mixture of peat moss and agrolite for hardening before transfer to soil. The Murashige and Skoog (Physiol. Plant. 15:473–497, 1962) mineral medium (MS) supplemented with 4.4 μM BA, 2.5 μM IBA, 11.7 μM AgNO_3y and 5.5% (w/v) sucrose gave most shoots, 8.9 μM BA, 2.5 μM IBA, 17.7 μM AgNO₃ and 5.5% (w/v) sucrose most roots, and 8.9 μM BA, 4.9 μM IBA, 11.7 μM AgNO₃ and 3.0% (w/v) sucrose most leaves, although other combinations were statistically equivalent in each case. Sucrose was the factor that most explained the variation in the promotion of shoots, roots, and leaves. The protocol developed resulted in up to 100% survival when plantlets were transferred to soil using AgNO₃, confirming that hardening of plantlets in vitro using hormonal stimulation was a suitable strategy to improve acclimatization.     

Growth of <Emphasis Type="Italic">Trametes versicolor</Emphasis> on phenol

Trametes versicolor 1 was shown to grow on phenol as its sole carbon and energy source. The culture growth and degradation ability dependence on culture medium pH value was observed. The optimal pH value of a liquid Czapek salt medium was 6.5. The investigated strain utilized completely 0.5 g/l phenol in 6 days. The dynamics of the phenol degradation process was investigated. The process was characterized by specific growth rate μ_max 0.33 h⁻¹, metabolic coefficient k = 4.4, yield coefficient Y _x/s  = 0.23 and rate of degradation Q = 0.506 h⁻¹. The intracellular activities of phenol hydroxylase (0.333 U/mg protein) and cis,cis-muconate lactonizing enzyme (0.41 U/mg protein) were demonstrated for the first time in this fungus. In an attempt to estimate the occurrence of gene sequences in T. versicolor 1 related to phenol degradation pathway a dot blot analysis with total DNA isolated from this strain was performed. Two synthetic oligonucleotides were used as hybridizing probes. One of the probes was homologous to the 5′end of phyA gene coding for phenol hydroxylase in Trichosporon cutaneum ATCC 46490. The other probe was created on the basis of cis,cis-muconate lactonizing enzyme coding gene in T. cutaneum ATCC 58094. The results of these investigations showed that T. versicolor 1 may carry genes similar to those of Trichosporon cutaneum capable to degrade phenol.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

<Emphasis Type="Italic">Catenaria anguillulae</Emphasis> Sorokin: a Natural Biocontrol Agent of <Emphasis Type="Italic">Meloidogyne graminicola</Emphasis> Causing Root Knot Disease of Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Summary Catenaria anguillulae parasitized and killed the eggs and second stage juveniles (J₂) of Meloidogyne graminicola under natural conditions. The percentage of infection in eggs was higher than J₂ of M.␣graminicola, which ranged between 0–50.3% and 0–18.9% in 2004 and 0–46.6% and 0–21.7% in 2005, respectively. The higher parasitism of eggs and J₂ was recorded from those fields in which plants were severely infected with M. graminicola. The degree of parasitism of eggs and J₂ by C. anguillulae varied with severity of root knot disease. The fields with a higher root gall index recorded a higher percentage of infection in eggs and J₂ of M. graminicola. In general, old galls when teased and incubated, recorded higher parasitism of eggs and juveniles than young galls.