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Bacteria capable of direct ammonia oxidation (Dirammox) play important roles in global nitrogen cycling and nutrient removal from wastewater. Dirammox process, NH3 → NH2OH → N2, first defined in Alcaligenes ammonioxydans HO‐1 and encoded by dnf gene cluster, has been found to widely exist in aquatic environments. However, because of multidrug resistance in Alcaligenes species, the key genes involved in the Dirammox pathway and the interaction between Dirammox process and the physiological state of Alcaligenes species remain unclear. In this work, ammonia removal via the redistribution of nitrogen between Dirammox and microbial growth in A. ammonioxydans HO‐1, a model organism of Alcaligenes species, was investigated. The dnfA, dnfB, dnfC, and dnfR genes were found to play important roles in the Dirammox process in A. ammonioxydans HO‐1, while dnfH, dnfG, and dnfD were not essential genes. Furthermore, an unexpected redistribution phenomenon for nitrogen between Dirammox and cell growth for ammonia removal in HO‐1 was revealed. After the disruption of the Dirammox in HO‐1, more consumed NH4+ was recovered as biomass‐N via rapid metabolic response and upregulated expression of genes associated with ammonia transport and assimilation, tricarboxylic acid cycle, sulfur metabolism, ribosome synthesis, and other molecular functions. These findings deepen our understanding of the molecular mechanisms for Dirammox process in the genus Alcaligenes and provide useful information about the application of Alcaligenes species for ammonia‐rich wastewater treatment. [ABSTRACT FROM AUTHOR]

Geobacter species, exhibiting exceptional extracellular electron transfer aptitude, hold great potential for applications in pollution remediation, bioenergy production, and natural elemental cycles. Nonetheless, a scarcity of well‐characterized genetic elements and gene expression tools constrains the effective and precise fine‐tuning of gene expression in Geobacter species, thereby limiting their applications. Here, we examined a suite of genetic elements and developed a new genetic editing tool in Geobacter sulfurreducens to enhance their pollutant conversion capacity. First, the performances of the widely used inducible promoters, constitutive promoters, and ribosomal binding sites (RBSs) elements in G. sulfurreducens were quantitatively evaluated. Also, six native promoters with superior expression levels than constitutive promoters were identified on the genome of G. sulfurreducens. Employing the characterized genetic elements, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was constructed in G. sulfurreducens to achieve the repression of an essential gene‐aroK and morphogenic genes‐ftsZ and mreB. Finally, applying the engineered strain to the reduction of tungsten trioxide (WO3), methyl orange (MO), and Cr(VI), We found that morphological elongation through ftsZ repression amplified the extracellular electron transfer proficiency of G. sulfurreducens and facilitated its contaminant transformation efficiency. These new systems provide rapid, versatile, and scalable tools poised to expedite advancements in Geobacter genomic engineering to favor environmental and other biotechnological applications. [ABSTRACT FROM AUTHOR]

Dissimilatory metal‐reducing bacteria (DMRB) can transfer electrons to extracellular insoluble electron acceptors and play important roles in geochemical cycling, biocorrosion, environmental remediation, and bioenergy generation. c‐type cytochromes (c‐Cyts) are synthesized by DMRB and usually transported to the cell surface to form modularized electron transport conduits through protein assembly, while some of them are released as extracellularly free‐moving electron carriers in growth to promote electron transport. However, the type of these released c‐Cyts, the timing of their release, and the functions they perform have not been unrevealed yet. In this work, after characterizing the types of c‐Cyts released by Geobacter sulfurreducens under a variety of cultivation conditions, we found that these c‐Cyts accumulated up to micromolar concentrations in the surrounding medium and conserved their chemical activities. Further studies demonstrated that the presence of c‐Cyts accelerated the process of microbial extracellular electron transfer and mediated long‐distance electron transfer. In particular, the presence of c‐Cyts promoted the microbial respiration and affected the physiological state of the microbial community. In addition, c‐Cyts were observed to be adsorbed on the surface of insoluble electron acceptors and modify electron acceptors. These results reveal the overlooked multiple roles of the released c‐Cyts in acting as public goods, delivering electrons, modifying electron acceptors, and even regulating bacterial community structure in natural and artificial environments. [ABSTRACT FROM AUTHOR]

Members of the genus Aeromonas prevail in aquatic habitats and have a great potential in biological wastewater treatment because of their unique extracellular electron transfer (EET) capabilities. However, the mediated EET mechanisms of Aeromonas have not been fully understood yet, hindering their applications in biological wastewater treatment processes. In this study, the electron shuttles in Aeromonas hydrophila, a model and widespread strain in aquatic environments and wastewater treatment plants, were explored. A. hydrophila was found to produce both flavins and 2‐amino‐3‐carboxy‐1,4‐naphthoquinone (ACNQ) as electron shuttles and utilize them to accelerate its EET for the bioreduction of various pollutants. The Mtr‐like respiratory pathway was essential for the reduction of flavins, but not involved in the ACNQ reduction. The electron shuttle activity of ACNQ for pollutant bioreduction involved the redox reactions that occurred inside the cell. These findings deepen our understanding about the underlying EET mechanisms in dissimilatory metal reducing bacteria and provide new insights into the roles of the genus Aeromonas in biological wastewater treatment. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis MR‐1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR‐1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base‐editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR‐1. The C‐to‐T conversion of suitable C within the base‐editing window could be readily and efficiently achieved by the pCBEso system without requiring double‐strand break or repair templates. Moreover, double‐locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N‐acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR‐1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild‐type when glucose or GlcNAc was used as the sole carbon source. Such a base‐editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR‐1 but also accelerates the robust construction of engineered strains for environmental bioremediation. [ABSTRACT FROM AUTHOR]

The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3′,5′‐monophosphate (cAMP) commonly exists in Shewanella strains and cAMP–cyclic adenosine 3′,5′‐monophosphate receptor protein (CRP) system regulates multiple bidirectional EET‐related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR‐1 (the strain MR‐1/pbPAC) and a MR‐1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR‐1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR‐1/pbPAC in the Cr(VI)‐reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP–CRP complex, which upregulates the expression levels of the genes encoding the c‐type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad‐spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity. [ABSTRACT FROM AUTHOR]

Dissimilatory metal reducer Geobacter sulfurreducens can mediate redox processes through extracellular electron transfer and exhibit potential‐dependent electrochemical activity in biofilm. Understanding the microbial acclimation to potential is of critical importance for developing robust electrochemically active biofilms and facilitating their environmental, geochemical, and energy applications. In this study, the metabolism and redox conduction behaviors of G. sulfurreducens biofilms developed at different potentials were explored. We found that electrochemical acclimation occurred at the initial hours of polarizing G. sulfurreducens cells to the potentials. Two mechanisms of acclimation were found, depending on the polarizing potential. In the mature biofilms, a low level of biosynthesis and a high level of catabolism were maintained at +0.2 V versus standard hydrogen electrode (SHE). The opposite results were observed at potentials higher than or equal to +0.4 V versus SHE. The potential also regulated the constitution of the electron transfer network by synthesizing more extracellular cytochrome c such as OmcS at 0.0 and +0.2 V and exhibited a better conductivity. These findings provide reasonable explanations for the mechanism governing the electrochemical respiration and activity in G. sulfurreducens biofilms. Acclimation of the electrochemically active microbe, Geobacter sulfurreducens, to potential level on microbial metabolism and electron transfer was described in this work. During biofilm formation on inert electrodes, optimized catabolism activity and cytochrome secretion are realized at +0.2 V than higher potentials. The observations and analysis uncovered findings on key factors for more efficient electron output by the electrochemically active biofilms, and suggested their values for engineering bioelectrochemical systems. [ABSTRACT FROM AUTHOR]

ABSTRACT Dinitrotoluene (DNT) is a widely present pollutant in aquatic environments, and its biodegradation is an economically attractive way to effectively removal. In aquatic environments, the presence of electrochemically active bacteria (EAB) could contribute to the anaerobic bioreduction of DNT. However, the mechanism behind such a biodegradation process at gene level remains to be further elucidated. In this work, the anaerobic reduction of 2,6-dinitrotoluene (2,6-DNT) by Shewanella oneidensis MR-1, a typical EAB in aquatic environments, was investigated. S. oneidensis MR-1 was found to be able to obtain energy for growth through the anaerobic respiration on 2,6-DNT. Experimental results show that the Mtr respiratory pathway, a transmembrane electron transport chain, was involved in the 2,6-DNT bioreduction. Knockout of cymA or nfnB resulted in a substantial loss of its 2,6-DNT-reducing ability, indicating that both CymA and NfnB were the key proteins in the microbial electron transfer chain. The genetic analysis further confirms that the Mtr respiratory pathway and NfnB are mainly responsible for the anaerobic reduction of 2,6-DNT by S. oneidensis MR-1. This work is useful to better understand the anaerobic bioreduction of nitroaromatic compounds in aquatic environments and remediate the environments contaminated by nitroaromatic compounds. Biotechnol. Bioeng. 2017;114: 761-768. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Cover Legend The cover image, by Dong‐Feng Liu et al., is based on the Article Anaerobic reduction of 2,6‐dinitrotoluene by Shewanella oneidensis MR‐1: Roles of Mtr respiratory pathway and NfnB, DOI: 10.1002/bit.26212. [ABSTRACT FROM AUTHOR]