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  • 1
    Electronic Resource
    Electronic Resource
    Studies of Host-Plasmid Interactions in Recombinant Microorganisms (1986)
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 469 (1986), S. 0 
    Details
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1749-6632.1986.tb26498.x
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  • 2
    Electronic Resource
    Electronic Resource
    Analysis of productivity in lysis-deficient lambda expression systems (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 40 (1992), S. 697-704 
    Details
    ISSN: 0006-3592
    Keywords: lambda phage ; lysogeny ; lytic state ; partial lysis ; multiplicity of infection ; temperature induction ; intracellular ; extracellular product ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The integrated state of λ in the host chromosome in lysogeny can be combined with its extrachromosomal replication in the lytic state to achieve high cloned gene productivities. Our previous studies on λ expression systems21,22 have shown 100% segregational stability of the cloned gene in lysogeny and cloned gene product levels up to 15% of total cell protein in a mutant lytic state. However, the expression phase of systems based on Escherichia coli JM109 and JM105 showed partial lysis of the productive culture despite a mutation in the lysis gene S of the lambda vector resulting in extracellular release of the cloned gene product. In the current study, we have eliminated partial lysis in the expression phase of λ systems and conducted a detailed comparative analysis of these systems in relation to maximization of cloned gene productivity. The elimination of partial cell lysis by using a nonpermissive strain Y1089 did not enhance product yields vs. earlier systems that exhibited partial lysis. The elimination of nonessential λ protein production by construction of a new vector NP326 did not yield higher product yields presumably because of the small fraction of these proteins in the lytic state. Temperature induction of the lysogen Y1089(NM1070) resulted in higher product levels than direct infection of Y1089 by the phage vector at a high multiplicity. Using infection experiments, we found the promoter lacUV5 in the vector λZEQS to yield threefold higher product levels than lac in NM1070, suggesting possible further enhancement of productivity with stronger promoters. The occurrence or absence of partial lysis in λ systems could be used beneficially to achieve extracellular or intracellular product as desired. The large capacity of λ vectors for insert DNA suggests potential applications in obtaining highly amplified levels of operons and multienzyme systems. © 1992 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260400608
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  • 3
    Electronic Resource
    Electronic Resource
    Mobilization of broad host range plasmid from Pseudomonas putida to established biofilm of Bacillus azotoformans. II. Modeling (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 280-286 
    Details
    ISSN: 0006-3592
    Keywords: biofilm ; plasmid transfer ; conjugation ; mathematical models ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A strain of Pseudomonas putida that harbors plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor. Transfer of the RK2 mobilizable pDLB101 plasmid to B. azotoformans was monitored over a 4-day period. Experimental results demonstrated that the broad host range, RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. In the companion article to this work, the rate of plasmid transfer was quantified as a function of the limiting nutrient, succinate, and as a function of the mechanism of transfer. A biofilm process simulation program (AQUASIM) was modified to analyze resultant experimental data. Although the AQUASIM package was not designed to simulate or predict genetic events in biofilms, modification of the rate process dynamics allowed successful modeling of plasmid transfer. For the narrow range of substrate concentrations used in these experiments, nutrient level had only a slight effect on the rate and extent of plasmid transfer in biofilms. However, further simulations using AQUASIM revealed that under nutrient poor conditions, the number of transconjugants appearing in the biofilm was limited. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 280-286, 1998.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Mobilization of broad host range plasmid from Pseudomonas putida to established biofilm of Bacillus azotoformans. I. Experiments (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 272-279 
    Details
    ISSN: 0006-3592
    Keywords: biofilm ; plasmid transfer ; conjugation ; retrotransfer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A strain of Pseudomonas putida harboring plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor under three different concentrations of the limiting nutrient, succinate. Experimental results demonstrated that the broad host range RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. At the lower concentrations, donor mediated plasmid transfer increased with increasing nutrient levels, but the highest nutrient concentration yielded the lowest rate of donor to recipient plasmid transfer. For transconjugant initiated transfer, the rate of transfer increased with increasing nutrient concentrations for all cases. At the lower nutrient concentrations, the frequency of plasmid transfer was higher between donors and recipients than between transconjugants and recipients. The reverse was true at the highest succinate concentration. The rates and frequencies of plasmid transfer by mobilization were compared to gene exchange by retrotransfer. The initial rate of retrotransfer was slower than mobilization, but then increased dramatically. Retrotransfer produced a plasmid transfer frequency more than an order of magnitude higher than simple mobilization. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 272-279, 1998.
    Additional Material: 7 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    Continuous culture dynamics for aniline metabolism by Pseudomonas sp. CIT1 (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 1-12 
    Details
    ISSN: 0006-3592
    Keywords: Pseudomonas ; substrate inhibition ; metabolic flux ; pathways analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Inhibition by toxic substrates enables multiple steady states to arise in biodegradation systems. This phenomenon was investigated for the continuous metabolism of aniline by Pseudomonas sp. CIT1. Differences of various metabolic parameters between the two growth regimes (uninhibited and inhibited) and the transient response to a step-up in dilution rate were determined. Regulatory mechanisms consistent with the experimental evidence are proposed.Aniline is the transcriptional inducer of a metabolic pathway that converts aniline to TCA cycle intermediates. The suite of enzymes is coordinately expressed from a single promoter. We followed the level of the pathway mRNA using a fragment containing the catechol 2,3 dioxygenase gene (andioxB) and monitored the pathway enzyme activity using catechol 2,3 dioxygenase (C23D). The inhibited regime resulted in a 60% lower growth yield, near constant levels of C23D monomer, but a 50% reduction in the specific activity of C23D, increased RNA synthesis rates (total and aniline pathway mRNA), and elevated RNA decay rates.Elucidation of regulatory mechanisms indicates that C23D is noncompetitively inhibited by aniline and subject to feedback inhibition by 2-hydroxymuconic semialdehyde (HMS). During uninhibited growth regime operation, metabolism of HMS is the rate-limiting step; in contrast, conversion of aniline to catechol limits growth in the inhibited regime. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:1-12, 1998.
    Additional Material: 15 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    Transient response simulations of recombinant microbial populations (1988)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 32 (1988), S. 418-429 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An asynchronous bacterial population has been approximated using a finite number of “computer” cells, each based on a complex single-cell model for Escherichia coli. This formulation correctly simulates the transient responses of protein and total cell mass synthesis rate to the sudden increase in the concentration of limiting energy source in the growth medium. Experimentally observed responses of rRNA and mRNA synthesis rates to growth rate shifts are qualitatively mirrored by the model. Simulation trends following those of a rel- mutant suggest that model modifications are needed to describe the dynamics of the stringent response. Simulations of the responses of recombinant populations to plasmid amplification or plasmid promoter induction also result in behavior similar to that determined experimentally. The calculated responses for recombinant populations subjected to constant promoter induction or cyclic induction-noninduction lead to the conclusion that inducible systems give greater productivity than those with fixed promoter strength. This formulation may be utilized as a basis for exploring other aspects of recombinant population dynamics.
    Additional Material: 12 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260320403
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  • 7
    Electronic Resource
    Electronic Resource
    Characterization of the mutant lytic state in lambda expression systems (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 369-377 
    Details
    ISSN: 0006-3592
    Keywords: lambda phage ; temperature-sensitive repressor ; amber mutations ; segregational stability ; lysogeny ; lytic state ; phage-host interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The two propagative phases of bacteriophage lambda, lysogeny and lysis, can be used in concert to enhance productivity of recombinant expression systems. Lambda vectors carrying mutations to prevent both cell lysis and λ DNA packaging in the lytic state have been shown to yield 100% stability of the product gene in lysogeny and to produce up to 15% of total cell protein as product β-galactosidase in a mutant lytic state.14 Despite these mutations, partial lysis of the culture was observed following induction of the cells from a lysogenic phase into the lytic state. To understand better the phage-host cell interactions and to investigate the possible cause(s) of lysis in these highly productive expression systems, we have made a detailed study of the suppressor-free system JM105(NM1070). We have found high levels of product (15% of total cell protein as β-glactosidase) to be due chiefly to a high-copy number of λ DNA in the mutant lytic state. There is partial lysis of the culture even in this suppressor-free system caused by a low-level natural suppression of the amber mutation in gene S of NM1070, resulting in accumulation of λ endolysin. We have also monitored changes in cell growth and morphology upon induction of the lysogen. There is a slight increase in cell number that follows a linear relationship with time and a 25-fold increase in cell volume during recombinat protein production in the mutant lytic state.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260390402
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  • 8
    Electronic Resource
    Electronic Resource
    Transcription from plasmid genes, macromolecular stability, and cell-specific productivity in Escherichia coli carrying copy number mutant plasmids (1989)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 34 (1989), S. 902-908 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Experiments were performed to evaluate, qualitatively and quantitatively, the adaptation of Escherichia coli to plasmid maintenance and cloned gene expression. Experimental findings indicate that the metabolic response to low plasmid levels is an increase of the biosynthetic capacity of both transcription and translation. At high copy number levels the gene-specific transcription rate continues to increase but the stability of plasmid-derived mRNA drops sharply. Protein levels are maintained, but translation efficiency decreases. These results indicate that cellular biosynthetic capacity may not be limiting productivity in recombinant systems. If macromolecular stability is the bottleneck, then current efforts to increase gene expression that focus on enhancing synthesis rates will be ineffective.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260340704
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