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Title: Final Technical Report: Genetic Control of Nitrogen Assimilation in Klebsiella oxytoca.

Abstract

Klebsiella oxytoca, an enterobacterium closely related to Escherichia coli and amenable to molecular genetic analysis, is a long-established model organism for studies of bacterial nitrogen assimilation. Our work concerned utilization of purines, nitrogen-rich compounds that are widespread in the biosphere. This project began with our observation that molybdenum cofactor (chlorate-resistant) mutants can use (hypo)xanthine as sole nitrogen source (Garzón et al., J. Bacteriol. 174:6298, 1992). Since xanthine dehydrogenase is a molybdoenzyme, Klebsiella must use an alternate route for (hypo)xanthine catabolsim. We identified and characterized a cluster of 22 genes that encode the enzymes, permeases and regulators for utilizing hypoxanthine and xanthine as sole nitrogen source. (Hypoxanthine and xanthine arise from deamination of adenine and guanine, respectively.) Growth and complementation tests with insertion mutants, combined with protein sequence comparisons, allow us to assign probable functions for the products of these genes and to deduce the overall pathway. We present genetic evidence that the first two enzymes for the Klebsiella purine utilization pathway have been recruited from pathways involved in catabolism of aromatic compounds. The first, HxaAB enzyme catalyzing (hypo)xanthine oxidation, is related to well-studied aromatic ring hydroxylating oxygenases such as phthalate dioxygenase. The second, HxbA enzyme catalyzing urate hydroxylation, is relatedmore » to single-component monooxygenases. Thus, the Klebsiella purine utilization pathway has likely experienced non-orthologous gene displacement, substituting these oxygenases for the conventional enzymes, xanthine dehydrogenase and uricase. We also present evidence that transcription of the hxaAB operon is subject to dual regulation: global general nitrogen regulation (Ntr) through an unknown mechanism, and (hypo)xanthine induction mediated by a LysR-type activator.« less

Authors:
Publication Date:
Research Org.:
UC-Davis
Sponsoring Org.:
USDOE - Office of Energy Research (ER)
OSTI Identifier:
900350
Report Number(s):
DOE/ER/20326
TRN: US200713%%271
DOE Contract Number:
FG03-99ER20326
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ADENINES; CATABOLISM; DEAMINATION; ENZYMES; ESCHERICHIA COLI; GENETIC CONTROL; HYPOXANTHINE; KLEBSIELLA; MOLYBDENUM; NITROGEN; NITRO-GROUP DEHYDROGENASES; OXIDATION; OXIDOREDUCTASES; OXYGENASES; PHTHALATES; XANTHINES; purine catabolism; hypoxanthine catabolism; xanthine catabolism; bacterial genetics; Klebsiella

Citation Formats

Valley Stewart. Final Technical Report: Genetic Control of Nitrogen Assimilation in Klebsiella oxytoca.. United States: N. p., 2007. Web. doi:10.2172/900350.
Valley Stewart. Final Technical Report: Genetic Control of Nitrogen Assimilation in Klebsiella oxytoca.. United States. doi:10.2172/900350.
Valley Stewart. Wed . "Final Technical Report: Genetic Control of Nitrogen Assimilation in Klebsiella oxytoca.". United States. doi:10.2172/900350. https://www.osti.gov/servlets/purl/900350.
@article{osti_900350,
title = {Final Technical Report: Genetic Control of Nitrogen Assimilation in Klebsiella oxytoca.},
author = {Valley Stewart},
abstractNote = {Klebsiella oxytoca, an enterobacterium closely related to Escherichia coli and amenable to molecular genetic analysis, is a long-established model organism for studies of bacterial nitrogen assimilation. Our work concerned utilization of purines, nitrogen-rich compounds that are widespread in the biosphere. This project began with our observation that molybdenum cofactor (chlorate-resistant) mutants can use (hypo)xanthine as sole nitrogen source (Garzón et al., J. Bacteriol. 174:6298, 1992). Since xanthine dehydrogenase is a molybdoenzyme, Klebsiella must use an alternate route for (hypo)xanthine catabolsim. We identified and characterized a cluster of 22 genes that encode the enzymes, permeases and regulators for utilizing hypoxanthine and xanthine as sole nitrogen source. (Hypoxanthine and xanthine arise from deamination of adenine and guanine, respectively.) Growth and complementation tests with insertion mutants, combined with protein sequence comparisons, allow us to assign probable functions for the products of these genes and to deduce the overall pathway. We present genetic evidence that the first two enzymes for the Klebsiella purine utilization pathway have been recruited from pathways involved in catabolism of aromatic compounds. The first, HxaAB enzyme catalyzing (hypo)xanthine oxidation, is related to well-studied aromatic ring hydroxylating oxygenases such as phthalate dioxygenase. The second, HxbA enzyme catalyzing urate hydroxylation, is related to single-component monooxygenases. Thus, the Klebsiella purine utilization pathway has likely experienced non-orthologous gene displacement, substituting these oxygenases for the conventional enzymes, xanthine dehydrogenase and uricase. We also present evidence that transcription of the hxaAB operon is subject to dual regulation: global general nitrogen regulation (Ntr) through an unknown mechanism, and (hypo)xanthine induction mediated by a LysR-type activator.},
doi = {10.2172/900350},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Wed Mar 07 00:00:00 EST 2007},
month = {Wed Mar 07 00:00:00 EST 2007}
}

Technical Report:

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  • Some microorganisms can use nitrate as the sole source of nitrogen for biosynthesis. This project focused on the bacterium Klebsiella oxytoca, an enterobacterium found in soil and water. Mutagenesis and molecular cloning identified the nasFEDCBA operon encoding enzymes for the uptake and reduction of nitrate and nitrite to ammonium, and the adjacent nasR regulatory gene. Analysis of nasF operon expression revealed that transcription is activated by the Ntr (general nitrogen regulation ) system in response to nitrogen limitation. Transcription antitermination control in response to nitrate and nitrite is mediated by the NasR protein. Additional work established that the NasR proteinmore » is an RNA-binding protein that interacts with nasF operon leader RNA to control transcription readthrough.« less
  • The current Verenium cellulosic ethanol process is based on the dilute-acid pretreatment of a biomass feedstock, followed by a two-stage fermentation of the pentose sugar-containing hydrolysate by a genetically modified ethanologenic Escherichia coli strain and a separate simultaneous saccharification-fermentation (SSF) of the cellulosic fraction by a genetically modified ethanologenic Klebsiella oxytoca strain and a fungal enzyme cocktail. In order to reduce unit operations and produce a fermentation beer with higher ethanol concentrations to reduce distillation costs, we have proposed to develop a simultaneous saccharification co-fermentation (SScF) process, where the fermentation of the pentose-containing hydrolysate and cellulosic fraction occurs within themore » same fermentation vessel. In order to accomplish this goal, improvements in the ethanologens must be made to address a number of issues that arise, including improved hydrolysate tolerance, co-fermentation of the pentose and hexose sugars and increased ethanol tolerance. Using a variety of approaches, including transcriptomics, strain adaptation, metagenomics and directed evolution, this work describes the efforts of a team of scientists from Verenium, University of Florida, Massachusetts Institute of Technology and Genomatica to improve the E. coli and K. oxytoca ethanologens to meet these requirements.« less
  • This economic assessment indicates that ammonia production by Klebsiella penumoniae is not economical with present strains; and improving nitrogen fixation to its theoretical limits in this organism is not sufficient to achieve economic viability. Contamination and reversion of the mutant are major technical problems. This leads to sterilization requirements which are economically prohibitive. Ammonia is a low value product and has been obtained only in dilute solutions with biological systems. Since the value of both the hydrogen produced by this organism and the methane value of the carbon source required greatly exceed the value of the ammonia formed, ammonia (fixedmore » nitrogen) should be considered the by-product and attention should be focused on other products. The production of hydrogen by Klebsiella or other anaerobic nitrogen fixers should receive additional study, since the value of hydrogen produced by Klebsiella greatly exceeds the value of the nitrogen fixed and since the activity of nitrogenase offers a significant improvement in hydrogen production. At observed efficiencies, the production of fixed nitrogen in the form of cell mass by Azotobacter is also uneconomical and the methane value of the carbon substrate exceeds the value of the nitrogen fixed. Parametric studies indicate that as efficiencies approach the theoretical limits the economics may become competititve under the assumptions of the economic model employed. The use of nif-derepressed microorganisms, particularly blue--green algae, may have significant potential for in situ fertilization in the environment. Additional work is required to determine: (1) the extent of in situ nitrogen fixation when nif-derepressed strains are added to the environment and; (2) how effective these strains are in increasing crop yields through the production of substances, other than fixed nitrogen, which may enhance plant growth.« less
  • Production of 2,3-butanediol by Klebsiella oxytoca is influenced by the degree of oxygen limitation. During batch culture studies, two phases of growth are observed: energy-coupled growth, during which cell growth and oxygen supply are coupled; and, energy-uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal 2,3-butanediol productivity occurs during the energy-coupled growth phase. In this article, a control system which maintains the batch culture at a constant level of oxygen limitation in the energy-coupled growth regime has been designed. Control, which involves feedback control on the oxygen transfer coefficient, is achieved by continually increasingmore » the partial pressure of oxygen in the feed gas, which in turn continually increases the oxygen transfer rate. Control has resulted in a balanced state of growth, a repression of ethanol formation, and an increase in 2,3-butanediol productivity of 18%.« less
  • The overall objective is to assess root physiological and morphological characteristics that may alter plant N acquisition capacity in response to rising atmospheric CO{sub 2} concentration. There is increasing evidence that plant and ecosystem responses to elevated levels of CO{sub 2} will ultimately depend on availability and acquisition rate of other resources such as N. Therefore, knowledge of any changes in root capacity to acquire N is crucial in predicting plant and ecosystem responses to high CO{sub 2}. Here the authors are testing two major hypotheses: (1) elevated CO{sub 2} will enhance root N uptake kinetics and (2) CO{sub 2}more » enrichment will increase root preference for NO{sub 3}{sup {minus}} as opposed to NH{sub 4}{sup +}. High CO{sub 2} enhances root energy status which should in turn favor energy-intensive processes such as NO{sub 3}{sup {minus}} uptake and assimilation. The above hypotheses are being tested on a range of species from native and agricultural ecosystems using a combination of field, lab and growth chamber studies. The authors have demonstrated a considerable interspecies variation in root N uptake responses to CO{sub 2} enrichment and attempts are now underway to evaluate if such variations are correlated with different functional groups. A comprehensive growth model, using physiological and allocation parameters, has been largely completed and will be used to analyze the completed experimental data.« less