Dynamics of Genomic-Library Enrichment and Identification of Solvent Tolerance Genes for Clostridium acetobutylicum

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA.
Applied and Environmental Microbiology (Impact Factor: 3.95). 06/2007; 73(9):3061-8. DOI: 10.1128/AEM.02296-06
Source: PubMed

ABSTRACT A Clostridium acetobutylicum ATCC 824 genomic library was constructed using randomly sheared DNA. Library inserts conferring increased tolerance to 1-butanol were isolated using two protocols. Protocol I utilized a single round of butanol challenges in batch culture, while protocol II, which gave clearly superior outcomes, was based on the serial transfer of stationary-phase cultures into progressively higher butanol concentrations. DNA microarray analysis made a high-resolution assessment of the dynamic process of library enrichment possible for the first time. Protocol I yielded a library insert containing the entire coding region of the gene CAC0003 (which codes for a protein of unknown function) but also several DNA fragments containing promoter regions. Protocol II enabled the successful identification of DNA fragments containing several intact genes conferring preferential growth under conditions of butanol stress. Since expression using the employed library is possible only from natural promoters, among the enriched genes, we identified 16 genes that constitute the first cistron of a transcriptional unit. These genes include four transcriptional regulators (CAC0977, CAC1463, CAC1869, and CAC2495). After subcloning plasmids carrying the CAC0003 and CAC1869 genes, strains 824(pCAC0003) and 824(pCAC1869) exhibited 13% and an 81% increases, respectively, in butanol tolerance relative to the plasmid control strain. 824(pCAC1869) consistently grew to higher cell densities in challenged and unchallenged cultures and exhibited prolonged metabolism. Our serial enrichment approach provided a more detailed understanding of the dynamic process of library enrichment under conditions of selective growth. Further characterization of the genes identified in this study will likely enhance our understanding of the complex phenotype of solvent tolerance.

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    • "Other growth kinetics parameters, ''percentage of tolerance'' and ''relative tolerance (RT)'', were calculated using Eqs. (4) and (5), respectively (Borden and Papoutsakis 2007). These parameters were calculated using the measured growth after a period of time. "
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    • "The same level of information is not available concerning anaerobic bacteria. Certain species such as Clostridium acetobutylicum and Zymomonas mobilis employed in the production of solvents and biofuels have been studied in detail (10, 45); however, very few data exist regarding the solvent tolerance of other anaerobic species important in CAH bioremediation settings. Because CAH-contaminated environments such as soil and aquifers are often complex and inhabited by a variety of species, it is useful to not only understand how solvents influence the species targeted as the catalyst of remediation but also co-existing species which may modulate bioremediation outcomes through mutualism or competition. "
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    ABSTRACT: The aim of this research was to evaluate the effects of four chlorinated aliphatic hydrocarbons (CAHs), perchloroethene (PCE), carbon tetrachloride (CT), chloroform (CF) and 1,2-dichloroethane (1,2-DCA), on the growth of eight anaerobic bacteria: four fermentative species (Escherichia coli, Klebsiella sp., Clostridium sp. and Paenibacillus sp.) and four respiring species (Pseudomonas aeruginosa, Geobacter sulfurreducens, Shewanella oneidensis and Desulfovibrio vulgaris). Effective concentrations of solvents which inhibited growth rates by 50% (EC50) were determined. The octanol-water partition coefficient or log Po/w of a CAH proved a generally satisfactory measure of its toxicity. Most species tolerated approximately 3-fold and 10-fold higher concentrations of the two relatively more polar CAHs CF and 1,2-DCA, respectively, than the two relatively less polar compounds PCE and CT. EC50 values correlated well with growth rates observed in solvent-free cultures, with fast-growing organisms displaying higher tolerance levels. Overall, fermentative bacteria were more tolerant to CAHs than respiring species, with iron- and sulfate-reducing bacteria in particular appearing highly sensitive to CAHs. These data extend the current understanding of the impact of CAHs on a range of anaerobic bacteria, which will benefit the field of bioremediation.
    Microbes and Environments 01/2014; 29(1). DOI:10.1264/jsme2.ME13113 · 2.42 Impact Factor
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    • "The relative growth rates were presented as the cell densities measured at a wavelength of 600 nm by spectrophotometer (GE Healthcare Life Sciences "GeneQuant 1300). Densities of toxin-treated cultures were normalized by the density of their respective toxin-free controls under otherwise same growth conditions [42]. From each tolerance assay, percent tolerance relative to unchallenged cultures was estimated at each challenge level and sample time as follows: "
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    ABSTRACT: Though n-butanol has been proposed as a potential transportation biofuel, its toxicity often causes oxidative stress in the host microorganism and is considered one of the bottlenecks preventing its efficient mass production. To relieve the oxidative stress in the host cell, metallothioneins (MTs), which are known as scavengers for reactive oxygen species (ROS), were engineered in E. coli hosts for both cytosolic and outer-membrane-targeted (osmoregulatory membrane protein OmpC fused) expression. Metallothioneins from human (HMT), mouse (MMT), and tilapia fish (TMT) were tested. The host strain expressing membrane-targeted TMT showed the greatest ability to reduce oxidative stresses induced by n-butanol, ethanol, furfural, hydroxymethylfurfural, and nickel. The same strain also allowed for an increased growth rate of recombinant E. coli under n-butanol stress. Further experiments indicated that the TMT-fused OmpC protein could not only function in ROS scavenging but also regulate either glycine betaine (GB) or glucose uptake via osmosis, and the dual functional fusion protein could contribute in an enhancement of the host microorganism's growth rate. The abilities of scavenging intracellular or extracellular ROS by these engineering E. coli were examined, and TMT show the best ability among three MTs. Additionally, the membrane-targeted fusion protein, OmpC-TMT, improved host tolerance up to 1.5% n-butanol above that of TMT which is only 1%. These results presented indicate potential novel approaches for engineering stress tolerant microorganism strains.
    Biotechnology for Biofuels 09/2013; 6(1):130. DOI:10.1186/1754-6834-6-130 · 6.22 Impact Factor
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