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Simultaneous Coexpression of Borrelia burgdorferi Erp Proteins Occurs through a Specific, erp Locus-Directed Regulatory Mechanism. Nazira El-Hage, 2002. Identification and Characterization of Assembly Proteins of CS5 Pili from Enterotoxigenic Escherichia coli. Thomas G. Duthy, 2002.This study investigated the role of three genes comprising part of the operon which encodes CS5 pili from enterotoxigenic Escherichia coli . In-frame gene deletions were constructed, and the effects on biogenesis of the pili were examined . A deletion in csfB abolished CsfA major subunit accumulation in the periplasm, which could be restored by trans-complementation with a complete copy of the csfB gene . Localization studies using an antibody against CsfB showed that this protein was periplasmically located, and thus CsfB is likely to function as the specific chaperone for CsfA . An in-frame deletion mutation in the csfE gene resulted in pili approximately three times longer than those of the wild-type strain, thereby indicating a role for CsfE in pilus length regulation . Localization studies using an antibody generated against CsfE showed low-level CsfE accumulation in the outer membranes . Modulation of csfE expression in trans did not reduce the mean length of the pilus below that of the wild type, which indicated that CsfE is not rate-limiting for termination of pilus assembly . Interestingly, a deletion in the csfF gene also resulted in an elongated pilus morphology identical to that of the csfE deletion strain . However, unlike CsfE, CsfF was shown to be rate-limiting for termination of assembly, since overexpression of CsfF in a csfF deletion strain resulted in a significant decrease in the mean length of the pilus compared to that of the wild type . When the same construct was introduced into the wild-type strain, pilus expression was abolished . Since CsfF bears significant homology to the proposed CsfB chaperone, CsfF was predicted to act as the specific chaperone for CsfE . A double deletion in the csfB and csfF genes was shown to abolish the periplasmic accumulation of both CsfA and CsfD pilins, which could be restored individually only when the strain was trans-complemented with a wild-type copy of csfB or csfF, respectively . Therefore, CsfF may chaperone not only CsfE but also CsfD . A model for CS5 biogenesis is also proposed based on these and previous observations . Biofilm-Specific Cross-Species Induction of Antimicrobial Compounds in Bacilli. Liming Yan, 2003.An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air . When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures . An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells . Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment . Release of these secondary metabolites was not due to the onset of sporulation . Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures . Neither glycerol nor ferric iron was required for production of these inducer compounds . Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10T and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B . licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10T and EI-25-8 could not . These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds . Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of Escherichia coli. Jörg Overhage, 2003.The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp . strain HR199, respectively . Resting cells of the corresponding recombinant strain E . coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter-1 . This biotransformation was scaled up to a 30-liter fermentation volume . The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture . The maximum concentration of ferulic acid obtained was 14.7 g liter-1 after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol . In a two-step biotransformation, E . coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E . coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J . Overhage, H . Priefert, and A . Steinbüchel, Appl . Environ . Microbiol . 65:4837-4847, 1999) . This process led to 0.3 g of vanillin liter-1, besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter-1 . The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp . strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E . coli XL1-Blue .
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