Omics technology draws a comprehensive heavy metal resistance strategy in bacteria.

The rapid industrial revolution significantly increased heavy metal pollution, becoming a major global environmental concern. This pollution is considered as one of the most harmful and toxic threats to all environmental components (air, soil, water, animals, and plants until reaching to human). Therefore, scientists try to find a promising and eco-friendly technique to solve this problem i.e., bacterial bioremediation. Various heavy metal resistance mechanisms were reported. Omics technologies can significantly improve our understanding of heavy metal resistant bacteria and their communities. They are a potent tool for investigating the adaptation processes of microbes in severe conditions. These omics methods provide unique benefits for investigating metabolic alterations, microbial diversity, and mechanisms of resistance of individual strains or communities to harsh conditions. Starting with genome sequencing which provides us with complete and comprehensive insight into the resistance mechanism of heavy metal resistant bacteria. Moreover, genome sequencing facilitates the opportunities to identify specific metal resistance genes, operons, and regulatory elements in the genomes of individual bacteria, understand the genetic mechanisms and variations responsible for heavy metal resistance within and between bacterial species in addition to the transcriptome, proteome that obtain the real expressed genes. Moreover, at the community level, metagenome, meta transcriptome and meta proteome participate in understanding the microbial interactive network potentially novel metabolic pathways, enzymes and gene species can all be found using these methods. This review presents the state of the art and anticipated developments in the use of omics technologies in the investigation of microbes used for heavy metal bioremediation.

Biodegradation of isoproturon by Escherichia coli expressing a Pseudomonas putida catechol 1,2-dioxygenase gene.

The phenylurea herbicides are persistent in soil and water, necessitating the creation of methods for removing them from the environment. This study aimed to examine the soil microbial diversity, searching for local bacterial isolates able to efficiently degrade the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea (IPU). The best isolates able to effectively degrade IPU were selected, characterized, and identified as Pseudomonas putida and Acinetobacter johnsonii. The catechol 1, 2-dioxygenase enzyme's catA gene was amplified, cloned, and expressed in E. coli M15. The Expressed E. coli showed high degradation efficiency (44.80%) as analyzed by HPLC after 15 days of inoculation in comparison to P. putida (21.60%). The expression of the catA gene in P. putida and expressed E. coli was measured using quantitative polymerase chain reaction (qPCR). The results displayed a significant increase in the mRNA levels of the catA gene by increasing the incubation time with IPU. Hydrophilic interaction chromatography (HILIC) mass spectrometry analysis revealed that three intermediate metabolites, 1-(4-isopropylphenyl)-3-methylurea (MDIPU), 4-Isopropylaniline (4-IA) and 1-(4-isopropylphenyl) urea (DDIPU) were generated by both P. putida and expressed E. coli. In addition, IPU-induced catA activity was detected in both P. putida and expressed E. coli. The supernatant of both P. putida and expressed E. coli had a significant influence on weed growth. The study clearly exhibited that P. putida and expressed E. coli were capable of metabolizing IPU influentially and thus could be utilized for bioremediation and biodegradation technology development.

Draft genome of Raoultella planticola, a high lead resistance bacterium from industrial wastewater.

Isolation of heavy metals-resistant bacteria from their original habitat is a crucial step in bioremediation. Six lead (Pb) resistant bacterial strains were isolated and identified utilizing 16S rRNA to be Enterobacter ludwigii FACU 4, Shigella flexneri FACU, Microbacterium paraoxydans FACU, Klebsiella pneumoniae subsp. pneumonia FACU, Raoultella planticola FACU 3 and Staphylococcus xylosus FACU. It was determined that all these strains had their Minimum inhibitory concentration (MIC) to be 2500 ppm except R. planticola FACU 3 has a higher maximum tolerance concentration (MTC) up to 2700 ppm. We evaluated the survival of all six strains on lead stress, the efficiency of biosorption and lead uptake. It was found that R. planticola FACU 3 is the highest MTC and S. xylosus FACU was the lowest MTC in this evaluation. Therefore, transmission electron microscopy (TEM) confirmed the difference between the morphological responses of these two strains to lead stress. These findings led to explore more about the genome of R. planticola FACU 3 using illumine Miseq technology. Draft genome sequence analysis revealed the genome size of 5,648,460 bp and G + C content 55.8% and identified 5526 CDS, 75 tRNA and 4 rRNA. Sequencing technology facilitated the identification of about 47 genes related to resistance to many heavy metals including lead, arsenic, zinc, mercury, nickel, silver and chromium of R. planticola FACU 3 strain. Moreover, genome sequencing identified plant growth-promoting genes (PGPGs) including indole acetic acid (IAA) production, phosphate solubilization, phenazine production, trehalose metabolism and 4-hydroxybenzoate production genes and a lot of antibiotic-resistant genes.

Draft genome analysis for Enterobacter kobei, a promising lead bioremediation bacterium.

Lead pollution of the environment poses a major global threat to the ecosystem. Bacterial bioremediation offers a promising alternative to traditional methods for removing these pollutants, that are often hindered by various limitations. Our research focused on isolating lead-resistant bacteria from industrial wastewater generated by heavily lead-containing industries. Eight lead-resistant strains were successfully isolated, and subsequently identified through molecular analysis. Among these, Enterobacter kobei FACU6 emerged as a particularly promising candidate, demonstrating an efficient lead removal rate of 83.4% and a remarkable lead absorption capacity of 571.9 mg/g dry weight. Furthermore, E. kobei FACU6 displayed a remarkable a maximum tolerance concentration (MTC) for lead reaching 3,000 mg/L. To further investigate the morphological changes in E. kobei FACU6 in response to lead exposure, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed. These analyses revealed significant lead adsorption and intracellular accumulation in treated bacteria in contrast to the control bacterium. Whole-genome sequencing was performed to gain deeper insights into E. kobei's lead resistance mechanisms. Structural annotation revealed a genome size of 4,856,454 bp, with a G + C content of 55.06%. The genome encodes 4,655 coding sequences (CDS), 75 tRNA genes, and 4 rRNA genes. Notably, genes associated with heavy metal resistance and their corresponding regulatory elements were identified within the genome. Furthermore, the expression levels of four specific heavy metal resistance genes were evaluated. Our findings revealed a statistically significant upregulation in gene expression under specific environmental conditions, including pH 7, temperature of 30°C, and high concentrations of heavy metals. The outstanding potential of E. kobei FACU6 as a source of diverse genes related to heavy metal resistance and plant growth promotion makes it a valuable candidate for developing safe and effective strategies for heavy metal disposal.

Multiplex CRISPR/Cas9-mediated genome editing to address drought tolerance in wheat.

Genome editing tools have rapidly been adopted by plant scientists for crop improvement. Genome editing using a multiplex sgRNA-CRISPR/Cas9 genome editing system is a useful technique for crop improvement in monocot species. In this study, we utilized precise gene editing techniques to generate wheat 3'(2'), 5'-bisphosphate nucleotidase (TaSal1) mutants using a multiplex sgRNA-CRISPR/Cas9 genome editing system. Five active TaSal1 homologous genes were found in the genome of Giza168 in addition to another apparently inactive gene on chromosome 4A. Three gRNAs were designed and used to target exons 4, 5 and 7 of the five wheat TaSal1 genes. Among the 120 Giza168 transgenic plants, 41 lines exhibited mutations and produced heritable TaSal1 mutations in the M1 progeny and 5 lines were full 5 gene knock-outs. These mutant plants exhibit a rolled-leaf phenotype in young leaves and bended stems, but there were no significant changes in the internode length and width, leaf morphology, and stem shape. Anatomical and scanning electron microscope studies of the young leaves of mutated TaSal1 lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. Sal1 mutant seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings. Our results indicate that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing is efficient tool for mutating more multiple TaSal1 loci in hexaploid wheat.

Gibberellic acid-induced hepatorenal dysfunction and oxidative stress: Mitigation by quercetin through modulation of antioxidant, anti-inflammatory, and antiapoptotic activities.

The plant growth regulator gibberellic acid (GA3) is widely used in agriculture in many countries. However, little is known about its danger to human health or its physiologic and biochemical pathways. Our study examined the effect of GA3 on liver and kidney function and the effect of quercetin on the hepatorenal toxicity induced by GA3 in four groups of male albino rats. For 4 weeks, the control group (CNT) received saline, the quercetin group (QR) received daily intraperitoneal injections of quercetin (50 mg/kg/BW) dissolved in saline, the gibberellic acid group (GA3) received GA3 (55 mg/kg/BW) via oral gavage, and the protective group (QR) was injected with quercetin and gavaged with GA3 in the same doses used in the QR and GA3 groups (50 mg/kg/BW +GA3 and 55 mg/kg/BW). GA3 induced liver and kidney injury, as shown by elevated serum glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and gamma-glutamyl transferase (GPT, GOT, and GGT) as well as increased levels of creatinine, urea, and uric acid. Hepatorenal toxicity was demonstrated by a significant increase in levels of serum and tissue malondialdehyde (MDA) and decreased antioxidant enzyme activity, such as catalase (CAT) and superoxide dismutase (SOD), accompanied by a subsequent decrease in glutathione peroxidase (GPx) levels in liver and kidney tissue of GA3-treated rats. Administration of quercetin (QR) significantly protected hepatorenal tissue against the toxic effect of GA3 through normalization of the hepatic and renal function markers. It also retrieved the antioxidant ability by modulating the hepatorenal toxic effect at the molecular level through upregulation of antiapoptotic genes and downregulation of transforming growth factor-ß1 (TFG-ß1), cyclooxygenase-2 (COX-2), and nuclear factor-kappa B (NF-κB). Impairment of liver and kidney function was confirmed by histologic and immunohistochemical analyses. Pretreatment with quercetin was effective at attenuating histopathologic changes in hepatic and renal tissues by regulating the immunoexpression of caspase-3 and Bcl-2 to return them to more normal values. PRACTICAL APPLICATIONS: The confirmed hepatorenal dysfunction caused by GA3 was ameliorated by quercetin administration. Moreover, quercetin demonstrated the potential to reverse hepatorenal dysfunction by regulating inflammatory and antioxidant properties, inhibiting the production of free radicals and inflammation-associated cytokines, and modulating antioxidants and antiapoptotic activity.

Overexpression of chalcone isomerase A gene in Astragalus trigonus for stimulating apigenin.

Apigenin is one of the most studied flavonoids and is widely distributed in the plant kingdom. Apigenin exerts important antioxidant, antibacterial, antifungal, antitumor activities, and anti-inflammatory effects in neurological or cardiovascular disease. Chalcone isomerase A (chiA) is an important enzyme of the flavonoid biosynthesis pathway. In order to enhance the apigenin production, the petunia chi A gene was transformed for Astragalus trigonus. Bialaphos survived plants were screened by PCR, dot blot hybridization and RT-PCR analysis. Also, jasmonic acid, salicylic acid, chitosan and yeast extract were tested to evaluate their capacity to work as elicitors for apigenin. Results showed that yeast extract was the best elicitor for induction of apigenin with an increase of 3.458 and 3.9 fold of the control for calli and cell suspension culture, respectively. Transformed cell suspension showed high apigenin content with a 20.17 fold increase compared to the control and 6.88 fold more than the yeast extract treatment. While, transformed T1 calli derived expressing chiA gene produced apigenin 4.2 fold more than the yeast extract treatment. It can be concluded that the highest accumulation of apigenin was obtained with chiA transgenic cell suspension system and it can be utilized to enhancement apigenin production in Astragalus trigonus.

Assessment of Agroeconomic Indicators of Sesamum indicum L. as Influenced by Application of Boron at Different Levels and Plant Growth Stages.

To achieve the nutritional target of human food, boron (B) has been described as an essential mineral in determining seed and theoretical oil yield of Sesamum indicum L. The research to increase its cultivation is garnering attention due to its high oil content, quality and its utilization for various purposes, which include human nutrition as well as its use in the food industry. For this, a two-year field experiment was performed at PAU, Punjab, India to determine the effect of different concentrations of foliar-applied B (20, 30 and 40 mg L-1) and different growth stages of crop, i.e., we measured the effects on agroeconomic indicators and certain quality parameters of sesame using different concentrations of B applied at the flowering and capsule formation stages as compared to using water spray and untreated plants. Water spray did not significantly affect the studied parameters. However, B application significantly increased the yield, uptake, antioxidant activity (AOA) and theoretical oil content (TOC) compared to those of untreated plants. The maximum increase in seed yield (26.75%), B seed and stover uptake (64.08% and 69.25%, respectively) as well as highest AOA (69.41%) and benefit to cost ratio (B:C ratio 2.63) was recorded when B was applied at 30 mg L-1 at the flowering and capsule formation stages. However, the maximum sesame yield and B uptake were recorded when B was applied at a rate of 30 mg L-1. A significant increase in TOC was also recorded with a B application rate of 30 mg L-1. For efficiency indices, the higher values of boron agronomic efficiency (BAE) and boron crop recovery efficiency (BCRE) were recorded when B was applied at 20 mg L-1 (5.25 and 30.56, respectively) and 30 mg L-1 (4.96 and 26.11, respectively) at the flowering and capsule formation stages. In conclusion, application of B @ 30 mg L-1 at the flowering and capsule formation stages seemed a viable technique to enhance yield, B uptake and economic returns of sesame.

Overexpression of bacterial katE gene improves the resistance of modified tomato plant against Fusarium oxysporum f. sp. lycopersici.

Tomato (Solanum lycopersicum L.) yield is severely affected by Fusarium fungal disease. To improve the resistance of tomato against Fusarium oxysporum f. sp. lycopersici (FOL), Escherichia coli katE gene was transformed into two tomato cultivars, namely Castle Rock and Super strain B, via Agrobacterium tumefaciens; the transformation efficiency was 5.6% and 3.5%, respectively. The integration of the katE gene into T0, T1, and T2 transgenic tomato lines was confirmed using PCR. In addition, DNA dot blot technique confirmed the integration of the katE gene into T2 transgenic tomato lines. The RT-PCR analysis confirmed that the katE gene could be expressed normally in the T2 modified lines. Under artificial infection with FOL, the non-modified plants exhibited more severe fungal disease symptoms than those observed in katE overexpression (OE) lines. Our analysis showed that the levels of three defense enzymes, namely superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were increased during transgenic T2 generation pre-treated with FOL. The bioassay of modified lines revealed that an average of 52.56% of the modified Castle Rock cultivar and 50.28% of the modified Super Strain B cultivar showed resistance under Fusarium infection. These results clearly indicate that the modified tomato plants, in which the katE gene was overexpressed, became more resistant to the infection by FOL than the wild-type plants. Our study has proven that the overexpression of the E. coli katE gene in the OE lines could be utilized to develop and improve the resistance against fungal diseases in the modified crops.

Improvement of sugarcane for borer resistance using Agrobacterium mediated transformation of cry1Ac gene.

The sugarcane (Saccharum X officinarum) is one of the most important crops used to produce sugar and raw material for biofuel in the world. One of the main causes for sucrose content and yield losses is the attack by insect. In this investigation, cry1Ac gene was introduced into sugarcane variety GT54-9(C9) using the Agrobacterium tumefaciens transformation method for transgenic sugarcane production presenting insect-resistance. The A. tumefaciens strain GV1303 including pARTcry1Ac vector was used for the production of transformed sugarcane. The Bacillus thuringiensis cry gene were successfully used to produce transgenic plants used for the improvement of both agronomic efficiency and product quality by acquiring insect resistance. PCR and Southern hybridization techniques were used to confirm the cry1Ac gene incorporation into sugarcane genome. Transformation percentage was 22.2% using PCR analysis with specific primers for cry1Ac and npt-II (Neomycin phosphotransferase) genes. The expression of cry1Ac gene was determined using reverse transcriptase polymerase chain reaction (RT-PCR), QuickStix test, and insect bioassays. Bioassays for transformed sugarcane plants showed high level of toxicity to Sesamia cretica giving 100% mortality of the larvae. Sugarcane insect resistance was improved significantly by using cry1Ac gene transformation.

Bacillus aryabhattai FACU: A promising bacterial strain capable of manipulate the glyphosate herbicide residues.

Glyphosate is a commonly used organophosphate herbicide that has an adverse impact on humans, mammals and soil microbial ecosystems. The redundant utilize of glyphosate to control weed growth cause the pollution of the soil environment by this chemical. The discharge of glyphosate in the agricultural drainage can also cause serious environmental damage and water pollution problems. Therefore, it is important to develop methods for enhancing glyphosate degradation in the soil through bioremediation. In this study, thirty bacterial isolates were selected from an agro-industrial zone located in Sadat City of Monufia Governorate, Egypt. The isolates were able to grow in LB medium supplemented with 7.2 mg/ml glyphosate. Ten isolates only had the ability to grow in a medium containing different concentrations of glyphosate (50, 100, 150, 200 and 250 mg/ml). The FACU3 bacterial isolate showed the highest CFU in the different concentrations of glyphosate. The FACU3 isolate was Gram-positive, spore-forming and rod-shape bacteria. Based on API 50 CHB/E medium kit, biochemical properties and 16S rRNA gene sequencing, the FACU3 isolate was identified as Bacillus aryabhattai. Different bioinformatics tools, including multiple sequence alignment (MSA), basic local alignment search tool (BLAST) and primer alignment, were used to design specific primers for goxB gene amplification and isolation. The goxB gene encodes FAD-dependent glyphosate oxidase enzyme that responsible for biodegradation process. The selected primers were successfully used to amplify the goxB gene from Bacillus aryabhattai FACU3. The results indicated that the Bacillus aryabhattai FACU3 can be utilized in glyphosate-contaminated environments for bioremediation. According to our knowledge, this is the first time to isolate of FAD-dependent glyphosate oxidase (goxB) gene from Bacillus aryabhattai.

Reduction of chromium-VI by chromium-resistant Escherichia coli FACU: a prospective bacterium for bioremediation.

The release of hexavalent chromium [Cr (VI)] into environments has resulted in many undesirable interactions with biological systems for its toxic potential and mutagenicity. Chromate reduction via chromium reductase (ChrR) is a key strategy for detoxifying Cr (VI) to trivalent species of no toxicity. In this study, ten bacterial isolates were isolated from heavily polluted soils, with a strain assigned as FACU, being the most efficient one able to reduce Cr (VI). FACU was identified as Escherichia coli based on morphological and 16S rRNA sequence analyses. Growth parameters and enzymatic actions of FACU were tested under different experimental conditions, in the presence of toxic chromium species. The E. coli FACU was able to reduce chromate at 100 µg/mL conceivably by reducing Cr (VI) into the less harmful Cr (III). Two distinctive optical spectroscopic techniques have been employed throughout the study. Laser-induced breakdown spectroscopy (LIBS) was utilized as qualitative analysis to demonstrate the presence of chromium with the distinctive spectral lines for bacteria such as Ca, Fe, and Na. While UV-visible spectroscopy was incorporated to confirm the reduction capabilities of E. coli after comparing Cr (III) spectrum to that of bacterial product spectrum and they were found to be identical. The chromate reductase specific activity was 361.33 µmol/L of Cr (VI) per min per mg protein. The FACU (EMCC 2289) 16S rRNA sequence and the ChrR-partially isolated gene were submitted to the DDBJ under acc. # numbers LC177419 and LC179020, respectively. The results support that FACU is a promising source of ChrR capable of bioremediation of toxic chromium species.

Identification and characterization of novel bacterial polyaromatic hydrocarbon-degrading enzymes as potential tools for cleaning up hydrocarbon pollutants from different environmental sources.

Polycyclic aromatic hydrocarbons (PAHs) are recalcitrant hazardous environmental contaminants. Various strategies, including chemical and physical like oxidation, fixation, leaching, and electrokinetic or biological-based techniques are used for remediation of polluted sites. Bioremediation of PAHs, via PAH-degrading endophytic and rhizospheric microbes, represent a time-/cost-effective way for ecorestoration. Four bacterial strains were isolated from contaminated soil on MSM supplemented with anthracene, alpha-naphthalene or catechol as sole carbon sources. These isolates were identified with 16S rRNA as Bacillus anthracis, B. cereus, B. mojavensis and B. subtilis. The degradation efficiency on the selected aromatic compounds was tested by HPLC analysis. B. subtilis showed the highest degradation efficiency of anthracene (99%) after five days of incubation. B. subtilis showed the highest catechol 1, 2 dioxygenase activity in MSM supplemented with anthracene. The enzyme was purified by gel filtration chromatography and characterized (70 kD, Km 2.7 µg and Vmax 178U/mg protein). The catechol 1,2 dioxygenase gene from the identified four bacterial strains were isolated and submitted to GenBank (accession numbers MG255165-MG255168). The gene expression level of catechol 1,2 dioxygenase was upregulated 23.2-fold during the 72 h of incubation period. Furthermore, B. subtilis is a promising strain to be used in bioremediation of aromatic compounds-contaminated environments.

Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for in vitro Culturing of the Plant Microbiota.

In order to improve the culturability and biomass production of rhizobacteria, we previously introduced plant-only-based culture media. We herein attempted to widen the scope of plant materials suitable for the preparation of plant-only-based culture media. We chemically analyzed the refuse of turfgrass, cactus, and clover. They were sufficiently rich to support good in vitro growth by rhizobacteria isolates representing Proteobacteria and Firmicutes. They were also adequate and efficient to produce a cell biomass in liquid batch cultures. These culture media were as sufficient as artificial culture media for the cultivation and recovery of the in situ rhizobacteria of barley (Hordeum murinum L.). Based on culture-dependent (CFU plate counting) and culture-independent analyses (qPCR), mowed turfgrass, in particular, supported the highest culturable population of barley endophytes, representing >16% of the total bacterial number quantified with qPCR. This accurately reflected the endophytic community composition, in terms of diversity indices (S', H', and D') based on PCR-DGGE, and clustered the plant culture media together with the qPCR root populations away from the artificial culture media. Despite the promiscuous nature of the plant materials tested to culture the plant microbiome, our results indicated that plant materials of a homologous nature to the tested host plant, at least at the family level, and/or of the same environment were more likely to be selected. Plant-only-based culture media require further refinements in order to provide selectivity for the in vitro growth of members of the plant microbiome, particularly difficult-to-culture bacteria. This will provide insights into their hidden roles in the environment and support future culturomic studies.

Boosting isoprene production via heterologous expression of the Kudzu isoprene synthase gene (kIspS) into Bacillus spp. cell factory.

Isoprene represents a key building block for the production of valuable materials such as latex, synthetic rubber or pharmaceutical precursors and serves as basis for advanced biofuel production. To enhance the production of the volatile natural hydrocarbon isoprene, released by plants, animals and bacteria, the Kudzu isoprene synthase (kIspS) gene has been heterologously expressed in Bacillus subtilis DSM 402 and Bacillus licheniformis DSM 13 using the pHT01 vector. As control, the heterologous expression of KIspS in E. coli BL21 (DE3) with the pET28b vector was used. Isoprene production was analyzed using Gas Chromatography Flame Ionization Detector. The highest isoprene production was observed by recombinant B. subtilis harboring the pHT01-kIspS plasmid which produced 1434.3 µg/L (1275 µg/L/OD) isoprene. This is threefold higher than the wild type which produced 388 µg/L (370 µg/L/OD) isoprene, when both incubated at 30 °C for 48 h and induced with 0.1 mM IPTG. Additionally, recombinant B. subtilis produced fivefold higher than the recombinant B. licheniformis, which produced 437.2 µg/L (249 µg/L/OD) isoprene when incubated at 37 °C for 48 h induced with 0.1 mM IPTG. This is the first report of optimized isoprene production in B. licheniformis. However, recombinant B. licheniformis showed less isoprene production. Therefore, recombinant B. subtilis is considered as a versatile host for heterologous production of isoprene.

Molecular identification and genetic diversity among Photorhabdus and Xenorhabdus isolates.

Five bacterial strains were isolated from the hemocoel of the greater wax moth larvae (Galleria mellonella) infected with the entomopathogenic nematodes: Heterorhabditis bacteriophora HP88, Heterorhabditis indicus RM1 and Heterorhabditis sp (S1), Steinernema abbasi and Steinernema sp. (S II). Strains were identified as Photorhabdus luminescens HRM1, P. luminescens HS1, P. luminescens HP88, Xenorhabdus indica and X. nematophila ATTC19061 using 16S rDNA sequence analysis. To reveal the genetic diversity among these strains, three molecular markers (RAPD, ISSR and SRAP) were employed. RAPD analysis showed 73.8 and 54.5 polymorphism percentages for the Photorhabdus and Xenorhabdus strains, respectively. ISSR analysis resulted in 70.1 and 75.2 polymorphism percentages among the Photorhabdus and Xenorhabdus strains, respectively. The SRAP analysis indicated that 75.6 and 61.2% genetic polymorphism was detected among Photorhabdus and Xenorhabdus strains, respectively. The cluster analysis grouped the three Photorhabdus strains together in one cluster and the two Xenorhabdus strains together in another cluster indicating the phylogenetic relationships among them. The genotype-specific markers detected from the three molecular markers (RAPD, ISSR and SRAP) were sufficient to distinguish between the different bacterial strains tested and can be used in the future IBM program that could be built on the use of these strains.

Expression of synthetic human tropoelastin (hTE) protein in Nicotiana tabacum.

Plant molecular farming (PMF) is an important growing prospective approach in plant biotechnology; it includes production of recombinant pharmaceutical and industrial proteins in large quantities from engineered plants. Elastin is a major protein component of tissues that require elasticity, it helps keep skin smooth as it stretches to allow normal. Elastin is used as a raw material for the cosmetic industry. In this work, we aimed to use plant as a bioreactor for the expression and production of the full human tropoelastin protein. Agrobacterium- mediated transient expression system into Nicotiana tabacum using syringe agroinfiltration was used to provide fast and convenient way to produce recombinant proteins with greater expression overall the plant leaf. This study aimed to establish an efficient and rapid system for transiently expression and production of human recombinant tropoelastin protein in transgenic N. tabacum plants. Modified elastin (ELN) gene was biosynthesized and cloned into pCambia1390 vector to be used into N. tabacum agroinfilteration. Optimization of codon usage for the human tropoelastin gene, without changing the primary structure of the protein was carried out to ensure high expression in tobacco plants. The obtained data proved that the 5(th) day post-infiltration is the optimum interval to obtain the maximum production of our recombinant protein. Southern blot analysis was able to detect 2175 bp fragment length representing the ELN orf (open reding frame). On the other hand, ELN -expression within plant's tissue was visualized by RT-PCR during the period 3-10 days post agroinfiltration. At the protein level, western and ELISA confirmed the expression of recombinant tropoelastin protein. Western blot analysis detected the tropoelastin protein as parent band at ∼70 kDa from freshly extracted protein, while two degraded bands of ∼55 and ∼45 kDa, representing a pattern of tropoelastin were appeared with frozen samples. This study showed that biosynthetic ELN gene was successfully expressed into N. tabacum leaves using agroinfiltration technique.