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A simple fed-batch process was carried out using constant and variable specific growth rates for high-cell-density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma(hIFN-gamma). The feeding rate was adjusted to achieve an appropriate specific growth rate. The dissolved oxygen level was maintained at 20-30% of air saturation by control of airflow and stirrer speed and, where necessary, by enrichment of inlet air with pure oxygen. Glucose was the sole source of carbon and energy and was provided by following a simple exponential feeding rate. The final cell density in the fed-batch fermentation with constant and variable specific growth rate feeding strategies was ~100 g dry cell wt l(-1) after 36 and 20 h, respectively. The final specific yield and overall productivity of recombinant hIFN-gamma in the variable specific growth rate strategy were 0.35 g rHu-IFN-gamma g(-1) dry cell wt and 0.9 g rHu-IFN-gamma l(-1) h(-1), respectively. A new chromatographic purification procedure involving anion exchange and cation exchange chromatographies was developed for purification of rHu-IFN-gamma from inclusion bodies. The established purification process is reproducible and the total recovery of rHu-IFN-gamma was ~30% (100 mg rHu-IFN-gamma g(-1) dry cell wt). The purity of the rHu-IFN-gamma was determined using HPLC. Sterility, pyrogenicity, and DNA content tests were conducted to assure the absence of toxic materials and other components of E. coli in the final product. The final purified rHu-IFN-gamma has a specific antiviral activity of ~2 x 10(7) IU/mg protein, as determined by viral cytopathic effect assay. These results certify the product for clinical purposes.

Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a yellow carotenoid pigment. A nonpigmented mutant was selected from a bank of mutants generated by random transposon mutagenesis. The transposon insertion site was mapped to the crtB gene, encoding a putative phytoene synthase, an enzyme involved in the early steps of carotenoid biosynthesis. We demonstrate here that the carotenoid pigment imparts protection against UV radiation and also contributes to the complete antioxidant pathway of P. stewartii. Moreover, production of this pigment is regulated by the EsaI/EsaR quorumsensing system and significantly contributes to the virulence of the pathogen in planta. P antoea stewartii subsp. stewartii (formerly Erwinia stewartii) is a yellow-pigmented, Gram-negative bacterial phytopathogen that causes a severe disease of sweet corn (Zea mays) called Stewart's wilt. The bacterium is introduced into the plant by its insect vector, the corn flea beetle (Chaetocnema pulicaria), where it colonizes both the apoplast and the xylem. Following systemic colonization of the plant, the bacteria exit the leaf tissue as a visible, yellow bacterial ooze. It is the preferential colonization of the xylem that blocks water flow in the plant and leads to the characteristic wilting associated with the disease. The type III secretion system, stewartan exopolysaccharide production, flagellum-based motility, and one host cell wall-degrading enzyme have been implicated as important pathogenicity or virulence factors for P. stewartii, but little is known about the biological role of the characteristic yellow pigment produced by P. stewartii (4,10,11,22,35).Carotenoids are among the most diverse natural products; they are synthesized by many organisms, including animals, plants, and microorganisms, and absorb light in the 400-to 550-nm range, which gives them their yellow-orange color (5). Several Erwinia species, which are close relatives of P. stewartii, produce yellow carotenoid pigments and possess a conserved carotenoid biosynthesis operon consisting of the genes crtE, crtX, crtY, crtI, and crtB in map order. Phytoene synthase, encoded by crtB, is the enzyme for the first step in carotenoid biogenesis, and mutations in crtB render a nonpigmented phenotype (36, 40, 52). More specifically, the P. stewartii genome contains a conserved carotenoid biosynthesis operon found in Erwinia spp., where crtE encodes geranylgeranyl pyrophosphate synthase, crtX encodes 3-hydroxy-␤-carotene glycosylase, crtY encodes lycopene cyclase, and crtI encodes phytoene dehydrogenase (43, 46). The crtB gene encodes phytoene synthase, which converts geranylgeranyl pyrophosphate to phytoene, an early step in the biosynthesis of ␤-carotene (41). Zeaxanthin diglucoside, a derivative of ␤-carotene, is the typical carotenoid produced by Erwinia spp. (2), and because of the high homology to the carotenoid biosynthetic operon in other Erwinia spp., we speculate that the P. stewartii likely produces zeaxanthin diglucoside or a cl...

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l )1 kanamycin and 200 mg l )1 cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and b-glucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T 1 plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. 'Western' immunoblot analysis of leaves isolated from T 0 transformants and progeny plants (T 1 ) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae. Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.

Erwinia amylovora, the causal agent of fire blight, carries the common plasmid pEA29 of 29 kb. To screen for occurrence of natural strains without plasmid pEA29, we applied PCR analysis with primers from the plasmid and the chromosomal ams region. In addition, a described TaqMan probe from pEA29 and newly designed primers from the amsregion were used for identification by real-time PCR. One strain isolated in Iran, one strain from Spain and two strains from Egypt lacked plasmid pEA29. From a recent screening series in southern Germany, in 123 E. amylovora strains from necrotic fire blight host plants, one strain was found without the common plasmid. The strains without pEA29 were virulent in assays with immature pears and on apple seedlings, but showed a reduced growth level in minimal medium without amino acids and thiamine. Transposon-labelled pEA29 was transformed into the plasmid-free strains resulting in restoration of this growth deficiency. The plasmid was stably maintained in these E. amylovora cells. The newly designed chromosomal primers for conventional and for realtime PCR identified E. amylovora strains in field samples lacking pEA29. These variants are apparently rare, but were detected in isolates from different regions in the world with fire blight.

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Iron is a key micronutrient for microbial growth but is often present in low concentrations or in biologically unavailable forms. Many microorganisms overcome this challenge by producing siderophores, which are ferric-iron chelating compounds that enable the solubilization and acquisition of iron in a bioactive form. Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a siderophore under iron-limiting conditions. The proteins involved in the biosynthesis and export of this siderophore are encoded by the iucABCD-iutA operon, which is homologous to the aerobactin biosynthetic gene cluster found in a number of enteric pathogens. Mutations in iucA and iutA resulted in a decrease in surfacebased motility that P. stewartii utilizes during the early stages of biofilm formation, indicating that active iron acquisition impacts surface motility for P. stewartii. Furthermore, bacterial movement in planta is also dependent on a functional siderophore biosynthesis and uptake pathway. Most notably, siderophore-mediated iron acquisition is required for full virulence in the sweet corn host, indicating that active iron acquisition is essential for pathogenic fitness for this important xylem-dwelling bacterial pathogen. P antoea stewartii subsp. stewartii, a Gram-negative bacterial phytopathogen, is the causal agent of a severe seedling wilt in susceptible corn cultivars called Stewart's wilt (1-3). The bacterium is transmitted by an insect vector, the corn flea beetle (Chaetocnema pulicaria), that introduces the pathogen into the plant tissue when it creates scratching wounds on the leaf surface during feeding. There are two phases of Stewart's wilt, a leaf blight and a wilting phase. During the initial infection phase, leaf blight occurs when the bacteria colonize the apoplastic space and cause characteristic water-soaked lesions. As infection progresses, the bacteria preferentially colonize the xylem, where they move systemically through the plant and block water flow, thereby causing the wilting observed in susceptible young seedlings. P. stewartii forms biofilms in the xylem, a process which is regulated in a cell density-dependent manner and requires flagellar-based surface motility and production of an exopolysaccharide (EPS) matrix (4-6). It is these biofilms and the associated EPS that presumably lead to xylem vessel blockage (5, 7). P. stewartii is able to reach very high cell densities and spread quickly within the xylem, in part due to flagellar motility and secretion of a plant cell wall-degrading enzyme (4, 8). However, it is not fully known how the bacterium obtains the suite of required nutrients for rapid growth within the plant or what environmental factors influence the transition from the planktonic to the biofilm state, which is necessary for systemic colonization of the xylem. Iron is commonly a limiting nutrient for microbial growth because it is often insoluble at biological pH within tissues and thus unavailable for utilization. Specifically, in plant tissues th...