Mechanism and Regulation of Organic and Heavy Metal Pollutants Transformation in Extracellular Respiratory Bacteria

胞外呼吸細菌轉化有機和重金屬污染物的機制解析和性能調控

Student thesis: Doctoral Thesis

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Author(s)

Related Research Unit(s)

Detail(s)

Awarding Institution
Supervisors/Advisors
  • T C LAU (Supervisor)
  • Wenwei LI (External person) (External Supervisor)
  • Guoping SHENG (External person) (External Supervisor)
Award date3 Jul 2020

Abstract

Extracellular respiratory bacteria (ERB) is a group of microorganisms capable of transferring the intracellular metabolic electrons to outside the cells. The extracellular electron transfer (EET) involve a stepwise electron movement from the inner-membrane, through the periplasm and outer-membrane and ultimately to the extracellular electron receptors. A wide range of extracellular electron receptors can be used by ERB, rendering them important roles in the geochemical cycling of many elements and environmental remediation. In addition, many ERB possess diverse EET pathways, which endows the bacteria powerful adaptability to the complicated and variable environment. However, the detailed EET mechanisms of ERB in pollutants transformation are largely unclear, which restrict the optimization such processes. In this thesis, we aim to clarify the mechanisms of EET and contaminants transformation by using several different ERB, and find effective regulation strategies for such processes. Our work sheds new light into the role of ERB in pollutant transformation and may a foundation for practical application of ERB in environmental remediation. The main research contents and results are as follows:

1. The extracellular respiratory pathways of Shewanella oneidensis MR-1 involved in the extracellular reduction of 2,6-dinitrotoluene (2,6-DNT) was elucidated. Our results confirm that S. oneidensis MR-1 could use 2,6-DNT as the sole electron acceptor to support its anaerobic respiration, and the 2,6-DNT reduction could be accelerated by adding riboflavin as electron mediators. The Mtr respiratory pathway and NfnB were identified as the key proteins involved in the extracellular reduction of 2,6-DNT. Product analysis showed that 2,6-DNT was eventually converted into 2,6-diaminotoluene, which can be easily degraded under aerobic conditions.

2. Synthetic biology approaches were applied to enhance the EET and extracellular pollutant degradation by Shewanella oneidensis MR‑1. Engineered bacteria with simultaneously enhanced flavin synthesis ability and metal reducing conduit was constructed. Specifically, the flavin biosynthesis gene cluster ribC-ribBA-ribE and metal-reducing conduit biosynthesis gene cluster mtrC-mtrA-mtrB were coexpressed in S. oneidensis MR-1 by means of synthetic biology. The engineered strain exhibited significantly strengthened EET ability and achieved 3-fold faster decolorization of methyl orange (MO) than the wide type strain. This work implies that synthetic biology approaches may be utilized as a powerful tool to further enhance the pollutant degradation ability of ERB and promote their practical environmental application.

3. The arsenic tolerance and the biomolecular pathways of reversible arsenic (As) redox transformation in Shewanella putrefaciens CN32 were elucidated. Based on quantitative PCR analysis of the gene transcription of arsenic resistance system (ars) and arsenic extracellular respiratory system (arr) in the bacterium under different arsenic induction conditions, we constructed several arsenic-related gene mutant strains by gene knockout. The results showed that arsDAB2C4 cluster contained the main genes involved in As(III) tolerance. The arsenic respiratory reductase Arr also could enhance the As(III) tolerance of the As(III)-sensitive strain E. coli AW3110. ArrAB was identified as the bidirectional enzyme that directly catalyzes the redox cycling of arsenic in vivo under aerobic conditions, which process is regulated by the supply of carbon substrate. This study deepens our understanding on the bacteria-mediated geochemical cycle of arsenic and may facilitate better control of arsenic pollutants in the environment.

4. A non-enzymatic dependent pathway of manganese (Mn) biomineralization by Shewanella putrefaciens CN32 was discovered. We found that S. putrefaciens CN32 could mineralize Mn(II) into Mn2O3, and stable Mn(III) was also formed in the reaction solution. Gene knockout experiments showed that laccase and siderophore were not involved in the oxidation of Mn(II), and a direct enzymatic oxidation of Mn(II) was excluded by enzyme inhibitors and heat treatment. The active factor for Mn(II) oxidation was mainly found in the supernatant, with molecular weight between 1 kDa and 3 kDa. The new Mn transformation pathway revealed in this work may facilitate a better understanding on the role of ERB in the biogeochemical cycling of Mn.

5. The EET pathway of another environmentally ubiquitous ERB, Aeromonas hydrophila ATCC7966, was deciphered. Our results showed that A. hydrophila could metabolize a wide range of carbon sources, and are capable of extracellular respiration via both direct and mediated EET pathway. A. hydrophila mainly uses the Mtr pathway for direct EET. In addition, it can secrete 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as an electron shuttle to further facilitate the EET. In view of the powerful ability of Aeromonas in degrading diverse environmental pollutants, this work implies a great potential of utilizing this bacteria for environmental remediation.

6. The dependency of arsenic resistance and reduction capacity of Aeromonas hydrophila on electron donor was elucidated. The genome analysis shows that the arsenic resistance system was widely found in the genus Aeromonas. The key role of the ars operon in determining the arsenic resistance was identified. The sensitivity of A. hydrophila to As(V) and As(III) and the As(V) reduction rate were found to depend heavily on the type of electron donor. Our results may facilitate a better understanding on the impacts of electron donors on the arsenic toxicity to microbes as well as the roles of the genus Aeromonas in the biogeochemical cycle of arsenic.

7. The mechanisms of biotransformation and immobilization of antimony (Sb) by soil-dwelling Pelosinus fermentans were elucidated. P. fermentans Ti_20, a novel member of the genus Pelosinus, was isolated from the Sb-contamination area and used for studying the Sb biotransformation. The Sb(V) reduction was mainly achieved through a two-electron transfer process, and Sb2O3 precipitates were identified as the main reduction product. The native-PAGE experiment showed that Sb(V) reductase is located in the periplasmic place of this strain. This study represents an important step in understanding the molecular mechanism of microbe-Sb interactions and the biogeochemical cycles of Sb in nature.

    Research areas

  • extracellular respiratory bacteria, extracellular electron transfer, 2,6-dinitrotoluene, manganese oxidation, azo dye, arsenic redox, antimony reduction