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.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

Shared molecular characteristics of successfully transformed mitochondrial genomes in <Emphasis Type="Italic">Chlamydomonas</Emphasis><Emphasis Type="Italic">reinhardtii</Emphasis>

Three types of respiratory deficient mitochondrial strains have been reported in Chlamydomonas reinhardtii: a deficiency due to (i) two base substitutions causing an amino acid change in the apocytochrome b (COB) gene (i.e., strain named dum-15), (ii) one base deletion in the COXI gene (dum-19), or (iii) a large deletion extending from the left terminus of the genome to somewhere in the COB gene (dum-1, -14, and -16). We found that these respiratory deficient strains of C. reinhardtii can be divided into two groups: strains that are constantly transformable and those could not be transformed in our experiments. All transformable mitochondrial strains were limited to the type that has a large deletion in the left arm of the genome. For these mitochondria, transformation was successful not only with purified intact mitochondrial genomes but also with DNA-constructs containing the compensating regions. In comparison, mitochondria of all the non-transformable strains have both of their genome termini intact, leading us to speculate that mitochondria lacking their left genome terminus have unstable genomes and might have a higher potential for recombination. Analysis of mitochondrial gene organization in the resulting respiratory active transformants was performed by DNA sequencing and restriction enzyme digestion. Such analysis showed that homologous recombination occurred at various regions between the mitochondrial genome and the artificial DNA-constructs. Further analysis by Southern hybridization showed that the wild-type genome rapidly replaces the respiratory deficient monomer and dimer mitochondrial genomes, while the E. coli vector region of the artificial DNA-construct likely does not remain in the mitochondria.     

Site-directed integration system using a combination of mutant <Emphasis Type="Italic">lox</Emphasis> sites for <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an optimal C. glutamicum genome, a precise site-directed gene integration method was developed by using a pair of mutant lox sites, one a right element (RE) mutant lox site and the other a left element (LE) mutant lox site. Two DNA fragments, 5.7 and 10.2 kb-long, were successfully integrated into the genome. The recombination efficiency of this system compared to that obtained by single crossover by homologous recombination was 2 orders of magnitude higher. Moreover, the integrated DNA remained stably maintained on removal of Cre recombinase. The Cre/mutant lox system thus represents a potentially attractive tool for integration of foreign DNA in the course of the engineering of C. glutamicum traits.     

Efficient authentic fine mapping of the rice blast resistance gene <Emphasis Type="Italic">Pik-h</Emphasis> in the <Emphasis Type="Italic">Pik</Emphasis> cluster,using new <Emphasis Type="Italic">Pik-h</Emphasis>-differentiating isolates

The Pik-h gene in rice confers resistance to several races of rice blast fungus (Magnaporthe oryzae), and has been classified as a member of the Pik cluster, one of the most resistance (R) gene-dense regions in the rice genome. However, the loss of a key mutant isolate has long made it difficult to differentiate Pik-h from other Pik group genes especially from Pik-m. We identified new natural isolates enabling the differentiation between Pik-h and Pik-m genes, and first confirmed the authenticity of the International Rice Research Institute (IRRI) “monogenic” line IRBLkh-K3, and then fine-mapped the Pik-h gene in the Pik cluster. Using 701 susceptible individuals among 3,060 siblings from a cross of IRBLkh-K3×CO39, the Pik-h region was delimited to 270 kb, the narrowest interval among the Pik group genes reported to date, in the cv. Nipponbare genome. Annotation of this genome region first revealed 6 NBS-LRR type R-gene analogs (RGAs), clustered within the central 120 kb, as possible counterparts of Pik-h and 6 other Pik group R genes. Interestingly, the Pik-h region and the cluster of RGAs were shown to be located 130 kb and 230 kb apart from Xa4 and Xa2 bacterial blight resistance genes, respectively, once classified as belonging to the Pik cluster. The closest recombination events were limited to the margins of the Pik-h region, and recombination was suppressed in the core interval with the RGA cluster. This fine-mapping, performed in a short time using an HEGS system, will facilitate utilization of the cluster’s genetic resources and help to elucidate the mechanism of evolution of R-genes. The presence of natural isolates also confirmed that evolution of Pik-h corresponds to pathogen evolution.     

Expression of the <Emphasis Type="BoldItalic">Escherichia coli pntAB</Emphasis> genes encoding a membrane-bound transhydrogenase in <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> improves <Emphasis Type="SmallCaps">l</Emphasis>-lysine formation

A critical factor in the biotechnological production of l-lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP⁺ via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysine-producing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by ∼10% on glucose, ∼70% on fructose, ∼50% on glucose plus fructose, and even by ∼300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type.     

Identification of new secreted proteins and secretion of heterologous amylase by <Emphasis Type="Italic">C. glutamicum</Emphasis>

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5–5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form α-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.     

Characterization of <Emphasis Type="Italic">EamaT1</Emphasis>, a member of <Emphasis Type="Italic">maT</Emphasis> family of transposable elements from the earthworm <Emphasis Type="Italic">Eisenia andrei</Emphasis> (Annelida,Oligochaeta)

The maT family is a unique clade within the Tc1-mariner superfamily, and their distribution is to date known as being limited to invertebrates. A novel transposon named EamaT1 is described from the genome of the earthworm Eisenia andrei. The full sized EamaT1 was obtained by degenerate and inverse PCR-based amplification. Sequence analysis of multiple copies of the EamaT1, which consisted of 0.9 and 1.4 kb elements, showed that the consensual EamaT1 with inverted terminal repeats (ITRs) of 69 bp was 1,422 bp long and flanked by a duplicated TA dinucleotide. The EamaT1 is present in approximately 120–250 copies per diploid genome but undergoes an inactivation process as a result of accumulating multiple mutations and is nonfunctional. The open reading frame (ORF) of the EamaT1 consensus encoding 356 amino acid sequences of transposase contained a DD37D signature and a conserved paired-like DNA binding motif for the transposition mechanism. The result of ITRs comparison confirmed their consensus terminal sequences (5′-CAGGGTG-3′) and AT-rich region on the internal bases for ITRs-transposase interaction.     

<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis>-based reporter genes for studying meristem development in poplar

We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended to stimulate meristem development to promote clonal propagation and genetic transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">MPK1</Emphasis> gene is required for filamentous growth induced by isoamyl alcohol in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from the alcoholic fermentation

The aim of this study was to evaluate the MPK1 (SLT2) gene deletion upon filamentous growth induced by isoamyl alcohol (IAA) in two haploid industrial strains of Saccharomyces cerevisiae using oligonucleotides especially designed for a laboratory S. cerevisiae strain. The gene deletion was performed by replacing part of the open reading frames from the target gene with the KanMX gene. The recombinant strains were selected by their resistance to G418, and after deletion confirmation by polymerase chain reaction, they were cultivated in a yeast extract peptone dextrose medium + 0.5% IAA to evaluate the filamentous growth in comparison to wild strains. Mpk1 derivatives were obtained for both industrial yeasts showing the feasibility of the oligonucleotides especially designed for a laboratory strain (Σ1278b) by Martinez-Anaya et al. (In yeast, the pseudohyphal phenotype induced by isoamyl alcohol results from the operation of the morphogenesis checkpoint. J Cell Sci 116:3423–3431, 2003). The filamentation rate in these derivatives was significantly lower for both strains, as induced by IAA. This drastic reduction in the filamentation ability in the deleted strains suggests that the gene MPK1 is required for IAA-induced filamentation response. The growth curves of wild and derivative strains did not differ substantially. It is not known yet whether the switch to filamentous growth affects the fermentative characteristics of the yeast or other physiological traits. A genetically modified strain for nonfilamentous growth would be useful for these studies, and the gene MPK1 could be a target gene. The feasibility of designed oligonucleotides for this deletion in industrial yeast strains is shown.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

Stable chloroplast transformation in cabbage (<Emphasis Type="Italic">Brassica oleracea </Emphasis>L. var. <Emphasis Type="Italic">capitata </Emphasis>L.) by particle bombardment

The objectives of this research were first to isolate plastid gene sequences from cabbage (Brassica oleracea L. var. capitata L.), and to establish the chloroplast transformation technology of Brassica. A universal transformation vector (pASCC201) for Brassica chloroplast was constructed with trnV–rrn16S (left) and trnI–trnA–rrn23S (right) of the IR_A region as a recombination site for the transformed gene. In transforming plasmid pASCC201, a chimeric aadA gene was cloned between the rrn16S and rrn23S plastid gene borders. Expression of aadA confers resistance to spectinomycin and streptomycin antibiotics. The uidA gene was also inserted into the pASCC201 and transferred into the leaf cells of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by 200 mg/l spectinomycin and streptomycin. After antibiotic selection, the regeneration percentage of the two cabbage cultivars was about 2.7–3.3%. The results of PCR testing and Southern blot analysis confirmed that the uidA and aadA genes were present in the chloroplast genome via homologously recombined. Northern blot hybridizations, immunoblotting and GUS histochemical assays indicated that the uidA gene were stable integrated into the chloroplast genome. Foreign protein was accumulated at 3.2–5.2% of the total soluble protein in transgenic mature leaves. These results suggest that the expression of a variety of foreign genes in the chloroplast genome will be a powerful tool for use in future studies.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Engineering <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for the production of pyruvate

A Corynebacterium glutamicum strain with inactivated pyruvate dehydrogenase complex and a deletion of the gene encoding the pyruvate:quinone oxidoreductase produces about 19 mM l-valine, 28 mM l-alanine and about 55 mM pyruvate from 150 mM glucose. Based on this double mutant C. glutamicum △aceE △pqo, we engineered C. glutamicum for efficient production of pyruvate from glucose by additional deletion of the ldhA gene encoding NAD⁺-dependent l-lactate dehydrogenase (LdhA) and introduction of a attenuated variant of the acetohydroxyacid synthase (△C–T IlvN). The latter modification abolished overflow metabolism towards l-valine and shifted the product spectrum to pyruvate production. In shake flasks, the resulting strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN produced about 190 mM pyruvate with a Y _P/S of 1.36 mol per mol of glucose; however, it still secreted significant amounts of l-alanine. Additional deletion of genes encoding the transaminases AlaT and AvtA reduced l-alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygen supply during growth and production (0–5% dissolved oxygen), the newly constructed strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN △alaT △avtA produced more than 500 mM pyruvate with a maximum yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol g_(CDW)⁻¹ h⁻¹ (i.e., 0.08 g g_(CDW) ⁻¹ h⁻¹) in the production phase.     

Anaerobic growth of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> using nitrate as a terminal electron acceptor

Corynebacterium glutamicum, a gram-positive soil bacterium, has been regarded as an aerobe because its growth by fermentative catabolism or by anaerobic respiration has, to this date, not been demonstrated. In this study, we report on the anaerobic growth of C. glutamicum in the presence of nitrate as a terminal electron acceptor. C. glutamicum strains R and ATCC13032 consumed nitrate and excreted nitrite during growth under anaerobic, but not aerobic, conditions. This was attributed to the presence of a narKGHJI gene cluster with high similarity to the Escherichia coli narK gene and narGHJI operon. The gene encodes a nitrate/nitrite transporter, whereas the operon encodes a respiratory nitrate reductase. Transposonal inactivation of C. glutamicum narG or narH resulted in mutants with impaired anaerobic growth on nitrate because of their inability to convert nitrate to nitrite. Further analysis revealed that in C. glutamicum, narK and narGHJI are cotranscribed as a single narKGHJI operon, the expression of which is activated under anaerobic conditions in the presence of nitrate. C. glutamicum is therefore a facultative anaerobe.     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.