The overall purpose of this project is to elucidate the way in which the synthesis of ethanol and related fermentation products are regulated in the facultative anaerobe Escherichia coli. In particular, the expression of the adhE gene which encodes the multifunctional alcohol dehydrogenase (AdhE), the key enzyme in fermentation, is being studied by means of bacterial genetics and molecular biology. The adhR gene, encoding a regulator which mediates the response of adhE to the NADH level, has been the main focus of our recent work on adhE regulation. We have demonstrated NADH-dependent binding of the AdhR protein to the adhE upstream region by gel retardation. Mutant versions of adhR, that allow expression of adhE even when NADH levels are low, have been isolated and will be further characterized. Other genes subject to AdhR regulation will be isolated and examined. In particular the adhB gene, which is controlled by both AdhR and Fnr, will be characterized, and its regulatory region dissected. In addition to AdhR/NADH, the expression of the adhE gene also responds to pyruvate levels, possibly via the PdhR regulatory protein. Under some culture conditions, E. coli switches from ethanol to succinate production. The effects on expression of adhE under such conditions are being analysed. Translation of the AdhE protein depends on processing of the adhE mRNA by ribonuclease III. We are investigating the requirements for this process. Another RNA-based phenomenon is the abolition of AdhE synthesis in certain tRNA modification mutants. The hypothesis that this effect is mediated via ribonuclease III is being tested. The results should contribute to our fundamental understanding of the genetic regulation of anaerobic growth. From a practical viewpoint, a deeper understanding of fermentation should help in the genetic engineering of more efficient producer organisms for any desired fermentation product.
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The objective of this project is to examine the regulation of lactic acid production by the facultative anaerobe Escherichia coli. In particular, the transcriptional and translational regulation of the ldhA gene which encodes the fermentative lactate dehydrogenase (LDH) will be studied by means of bacterial genetics and molecular biology. The ldhA gene is induced by low pH, but only under anaerobic conditions. It is not regulated by the fnr gene which controls many genes involved in anaerobic respiration, nor by the adhR gene which regulates alcohol fermentation, nor, for that matter, by any other characterized regulatory system. The ldhA gene has been cloned and sequenced. We have constructed gene fusions of the ldhA regulatory region to both cat and lacZ and are using these to investigate how the expression of ldhA is regulated. Primer extension will be used to define the start of transcription of ldhA. We are using a PCR approach to dissect the upstream regulatory region of the ldhA gene in order to more precisely locate both those sites required for expression and those involved in regulation, including possible translational effects. In addition we will search for the presumed regulatory genes which control ldhA. Both insertion mutagenesis and physiologically based selections will be used to isolate mutations in novel regulatory genes which affect expression of the ldhA-lacZ fusions. The mutations will be genetically mapped and the genes characterized. We will also attempt to analyse the signal which elicits ldhA induction. Is it internal pH or external pH? Is it accumulation of one particular acidic product or a general response to pH? - and so forth. The stationary phase induction of ldhA will be similarly investigated. The results should contribute to our fundamental understanding of fermentation and its genetic regulation. From a practical viewpoint, a deeper understanding of fermentation should help in the genetic engineering of more efficient producer organisms for any desired fermentation product.
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The adhB mutation results in a decrease of upto 80% in the alcohol dehydrogenase depending on the growth conditions and the strain used. This is why we named it adhB; however, we now think this effect on ADH is probably indirect. In some strains it prevents anaerobic growth on minimal glucose medium (and other sugars). The adhB gene is regulated by the adhR gene (which also regulates the adhE gene encoding the alcohol dehydrogenase). The product of the adhB gene is still unknown, but the best guess (from map location and DNA homologies) is that it is involved in nitrogen metabolism anaerobically - perhaps an asparaginase or aspartase. The adhB gene is induced anaerobically but only in rich medium and is repressed by glucose. Both the Crp and Fnr regulatory proteins affect expression of adhB.
E. coli strain NZN111 (ldhA::Kan pfl::Cam) is unable to ferment and cannot grow anaerobically on sugars due to lacking both pyruvate formate lyase and lactate dehydrogenase. Strain AFP111 is a spontaneous mutant of NZN111 that regained the ability to grow anaerobically. AFP111 ferments glucose to a mixture of succinic acid, acetic acid and ethanol in a 2:1:1 ratio. In addition to this novel distribution of fermentation products, AFP111 also differs from its parental stain in that it lacks normal catabolite repression. The chromosomal mutation in AFP111 is in the ptsG gene, which encodes the membrane component of the glucose specific phosphoenolpyruvate: phospho-transferase system. Disruption of the ptsG gene in various strains in the parental lineage of AFP111 resulted in both a loss of normal catabolite repression and the production of succinic acid as the major fermentation product from glucose. These results indicate that the metabolic pathway to succinic acid is subject to catabolite repression in the presence of a functional ptsG gene and that mutation of ptsG results in increased production of succinic acid.
Since AFP111 still makes some ethanol and acetate it must convert some pyruvate to acetyl-CoA somehow. It needs some acetyl-CoA as a metabolic precursor. AFP111 lacks the normal pyruvate formate lyase (DEpfl::Cam). We have eliminated the pyruvate dehydrogense (aceEF), the pyruvate oxidase (poxB), the PFL of the anaerobic threonine degradation system (tdcE) and the pyruvate ferredoxin oxidoreductase (fdx::Kan). This leaves the pflCD operon and yfiD which according to their sequences code for extra PFLs. Whether and when these genes are functional or what their real role may be is unknown.
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Genes for Biodegradation of Aromatic Sulfones & Sulfonic Acids
In collaboration with Dr. Brian Klubek, Department of Plant & Soil Science, SIU.
Our system consists of the synergistic use of Corynebacterium strain 26 and Moraxella strain 16. The Corynebacterium can degrade aromatic sulfoxides or sulfones and the Moraxella degrades aromatic sulfonic acids. Together they can fully degrade dibenzothiophene sulfone, releasing essentially 100% of the sulfur as sulfite (spontaneously oxidised by air to sulfate) and generating intermediates characteristic of the "angular oxidation" or Brevibacterium pathway.
The Corynebacterium or Brevibacterium pathway is apparently intended to degrade a range of polycyclic aromatics as carbon and energy sources and sulfur elimination is incidental. Dihydroxy-biphenyl is the first sulfur free compound. Hence this pathway proceeds at a much higher rate than the Rhodococcus pathway, which is apparently designed for sulfur scavenging, and yields hydroxy-biphenyl. Our Moraxella isolate scarcely grows on benzene sulfonate etc., although it releases inorganic sulfur efficiently from aromatic sulfonates. When the Corynebacterium is also present, it produces breakdown products from the aromatic skeletons and so cross-feeds the Moraxella. Together, both grow and desulfurize much more effectively.
We intend to clone the Corynebacterium genes for angular oxidation of DBT sulfone and the Moraxella genes for aromatic desulfonation. These will be combined in a single host, possibly a pseudomonad, for expression of rapid DBT sulfone degrading activity. Our approach for the Corynebacterium will be to make mutants incapable of growing on DBT sulfone or related compounds and use these as hosts to clone intact copies of the various dbs genes. We have already isolated several mutants with blocks at different places in the DBT sulfone pathway in a close relative. Corynebacterium strain 2 has the same pathway, although it grows slower, than the later isolate, strain 26. A gene library from strain 26 will be used as source of the dbs genes and either our present mutants, or a new series in strain 26 itself, will be used to select for recovery of growth on DBT sulfone. A Corynebacterium genetic system is available. Plasmids carrying cloned DNA are transferred into Corynebacterium by conjugation with E. coli carrying the both the vector and the promiscuous RP1 plasmid integrated into its chromosome. Although the vector cannot replicate in Corynebacterium, it survives long enough for recombination with the chromosome to occur. This allows direct selection of genes which complement growth defects in Corynebacterium and, of course, their genetic manipulation in E. coli.
Little is known of the molecular biology of Moraxella. Consequently we will use a different approach. Desulfonation will detoxify aryl sulfonate detergents. We will use a gene library from Moraxella carried on an E. coli plasmid and will select for gain of resistance to alkyl benzene sulfonate detergents by E. coli strains which contain the plasmids. Another possibility is the use of indicator dyes, many of which are naphthalene or anthraquinone sulfonate derivatives. Loss of sulfonate groups will change the color of the dye, allowing visual screening. Eventually, we would like to get a cloning system for Moraxella, if possible. Moraxella is related, albeit not closely, to Neisseria and Acinetobacter, both of which have been investigated moderately well and have cloning systems available. It is possible that plasmids able to replicate in either of these organisms might work for Moraxella.
Biodegradation of sulfonate detergents is of interest in its own right. The ability to degrade alkyl sulfates, such as sodium dodecyl sulfate, is quite widespread, and is present in several Pseudomonas strains in our collection. Constructing a strain able to degrade both alkyl sulfate and aryl sulfonate based detergents might be a useful future project.
This project is presently unfunded.
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SIUC / College of Science / Microbiology / clark/
http://www.micro.siu.edu/clark/research.html
Last updated: 4-Aug-99 / dc