Evidence for quinol oxidase activity of ImoA, a novel NapC/NirT family protein from the neutrophilic Fe(II) oxidizing bacterium Sideroxydans lithotrophicus ES-1

The freshwater chemolithoautotrophic Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes Fe(II) at the cell surface. In this organism, it is proposed that the monoheme cytochrome MtoD from the Mto pathway transfer electrons across the periplasm to an inner membrane NapC/NirT family tetraheme cytochrome encoded by Slit_2495, for which we propose the name ImoA (inner membrane oxidoreductase). ImoA has been proposed to function as the quinone reductase, receiving electrons from iron oxidizing extracellular electron uptake pathway to reduce the quinone pool. In this study, ImoA was cloned on a pBAD plasmid vector and overexpressed in Escherichia coli. Biochemical and spectroscopic characterization of the purified ImoA reveals that this 26.5 kDa cytochrome contains one high-spin and three low-spin hemes. Our data show that ImoA can function as a quinol oxidase and is able to functionally replace CymA, a related NapC/NirT family tetraheme cytochrome required for anaerobic respiration of a wide range of substrates by Shewanella oneidensis. We demonstrate that ImoA can transfer electrons to different periplasmic proteins from S. oneidensis including STC and FccA, but in a manner that is distinct from that of CymA. Phylogenetic analysis shows that ImoA is clustered closer to NirT sequences than to CymA. This study suggests that ImoA functions as a quinol oxidase in S. oneidensis and raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus ES-1.

Biotic and Abiotic Biostimulation for the Reduction of Hexavalent Chromium in Contaminated Aquifers

Hexavalent chromium is a carcinogenic heavy metal that needs to be removed effectively from polluted aquifers in order to protect public health and the environment. This work aims to evaluate the reduction of Cr(VI) to Cr(III) in a contaminated aquifer through the stimulation of indigenous microbial communities with the addition of reductive agents. Soil-column experiments were conducted in the absence of oxygen and at hexavalent chromium (Cr(VI)) groundwater concentrations in the 1000–2000 μg/L range. Two carbon sources (molasses and EVO) and one iron electron donor (FeSO4·7H2O) were used as ways to stimulate the metabolism and proliferation of Cr(VI) reducing bacteria in-situ. The obtained results indicate that microbial anaerobic respiration and electron transfer can be fundamental to alleviate polluted groundwater from hazardous Cr(VI). The addition of organic electron donors increased significantly Cr(VI) reduction rates in comparison to natural soil attenuation rates. Furthermore, a combination of organic carbon and iron electron donors led to a longer life span of the remediation process and thus increased total Cr(VI) removal. This is the first study to investigate biotic and abiotic Cr(VI) removal by conducting experiments with natural soil and by applying biostimulation to modify the natural existing microbial communities.

Self-activating anti-infection implant

Clinically, it is difficult to endow implants with excellent osteogenic ability and antibacterial activity simultaneously. Herein, the self-activating implants modified with hydroxyapatite (HA)/MoS2 coating are designed to prevent Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infections and accelerate bone regeneration simultaneously. The electron transfer between bacteria and HA/MoS2 is triggered when bacteria contacted with the material. RNA sequencing data reveals that the expression level of anaerobic respiration–related genes is up-regulated and the expression level of aerobic respiration–related genes is down-regulated when bacteria adhere to the implants. HA/MoS2 presents a highly effective antibacterial efficacy against both S. aureus and E. coli because of bacterial respiration–activated metabolic pathway changes. Meanwhile, this coating promotes the osteoblastic differentiation of mesenchymal stem cells by altering the potentials of cell membrane and mitochondrial membrane. The proposed strategy exhibits great potential to endow implants with self-activating anti-infection performance and osteogenic ability simultaneously.

Aerobic Bacteria Produce Nitric Oxide via Denitrification and Trigger Algal Population Collapse

Microbial interactions govern marine biogeochemistry. These interactions are generally considered to rely on exchange of organic molecules. Here we report on a novel inorganic route of microbial communication, showing that algal-bacterial interactions are mediated through inorganic nitrogen exchange. Under oxygen-rich conditions, aerobic bacteria reduce algal-secreted nitrite to nitric oxide (NO) through denitrification, a well-studied anaerobic respiration mechanism. Bacteria secrete NO, triggering a cascade in algae akin to programmed cell death. During death, algae further generate NO, thereby propagating the signal in the algal population. Eventually, the algal population collapses, similar to the sudden demise of oceanic algal blooms. Our study suggests that the exchange of denitrification intermediates, particularly in oxygenated environments, is an overlooked yet ecologically significant route of microbial communication within and across kingdoms.

Oxidative respiration through the bd-I and cbb3 oxidases is required for Vibrio cholerae pathogenicity and proliferation in vivo.

Vibrio cholerae respires both aerobically and anaerobically and, while oxygen may be available to it during infection, other terminal electron acceptors are proposed for population expansion during infection. Unlike gastrointestinal pathogens that stimulate significant inflammation leading to elevated levels of oxygen or alternative terminal electron acceptors, V. cholerae infections are not understood to induce a notable inflammatory response. To ascertain the respiration requirements of V. cholerae during infection, we used Multiplex Genome Editing by Natural Transformation (MuGENT) to create V. cholerae strains lacking aerobic or anaerobic respiration. V. cholerae strains lacking aerobic respiration were attenuated in infant mice 10 5 -fold relative to wild type, while strains lacking anaerobic respiration had no colonization defect, contrary to earlier work suggesting a role for anaerobic respiration during infection. Using several approaches, including one we developed for this work termed Comparative Multiplex PCR Amplicon Sequencing (CoMPAS), we determined that the bd-I and cbb3 oxidases are essential for small intestinal colonization of V. cholerae in the infant mouse. The bd-I oxidase was also determined as the primary oxidase during growth outside the host, making V. cholerae the only example of a Gram-negative bacterial pathogen in which a bd-type oxidase is the primary oxidase for energy acquisition inside and outside of a host.

Loss of the acetate switch in Vibrio vulnificus enhances predation defence against Tetrahymena pyriformis

Vibrio vulnificus is an opportunistic human pathogen and autochthonous inhabitant of coastal marine environments, where the bacterium is under constant predation by heterotrophic protists or protozoans. As a result of this selection pressure, genetic variants with anti-predation mechanisms are selected for and persist in the environment. Such natural variants may also be pathogenic to animal or human hosts, making it important to understand these defence mechanisms. To identify anti-predator strategies, thirteen V. vulnificus strains of different genotypes isolated from diverse environments were exposed to predation by the ciliated protozoan, Tetrahymena pyriformis , and only strain ENV1 was resistant to predation. Further investigation of the cell-free supernatant showed that ENV1 acidifies the environment by the excretion of organic acids, which is toxic to T. pyriformis . As this predation resistance was dependent on the availability of iron, transcriptomes of V. vulnificus in iron-replete and iron-deplete conditions were compared. This analysis revealed that ENV1 ferments pyruvate and the resultant acetyl-CoA leads to acetate synthesis under aerobic conditions, a hallmark of overflow metabolism. The anaerobic respiration global regulator, arcA , was upregulated when iron was available. An Δ arcA deletion mutant of ENV1 accumulated less acetate and importantly, was sensitive to grazing by T. pyriformis . Based on the transcriptome response and quantification of metabolites, we conclude that ENV1 has adapted to overflow metabolism and has lost a control switch that shifts metabolism from acetate excretion to acetate assimilation, enabling it to excrete acetate continuously. We show that overflow metabolism and the acetate switch contribute to prey-predator interactions. Importance Bacteria in the environment, including Vibrio spp., interact with protozoan predators. To defend against predation, bacteria evolve anti-predator mechanisms ranging from changing morphology, biofilm formation and secretion of toxins or virulence factors. Some of these adaptations may result in strains that are pathogenic to humans. Therefore, it is important to study predator defence strategies of environmental bacteria. V. vulnificus thrives in coastal waters and infects humans. Very little is know about the defence mechanisms V. vulnificus expresses against predation. Here we show that a V. vulnificus strain (ENV1) has rewired the central carbon metabolism enabling the production of excess organic acid that is toxic to the protozoan predator, T. pyriformis . This is a previously unknown mechanism of predation defence that protects against protozoan predators.

Metabolic Potential of Microbial Communities in the Hypersaline Sediments of the Bonneville Salt Flats

The Bonneville Salt Flats (BSF) appear to be entirely desolate when viewed from above, but in reality they host rich microbial communities just below the surface salt crust. In this study, we investigate the metabolic potential of the BSF microbial ecosystem. The predicted and measured metabolic activities provide new insights into the ecosystem functions of evaporite landscapes and are an important analog for potential subsurface microbial ecosystems on ancient and modern Mars. Hypersaline and evaporite systems have been investigated previously as astrobiological analogs for Mars and other salty celestial bodies. Still, these studies have generally focused on aquatic systems and cultivation-dependent approaches. Here, we present an ecosystem-level examination of metabolic pathways within the shallow subsurface of evaporites. We detected aerobic and anaerobic respiration as well as methanogenesis in BSF sediments. Metagenome-assembled genomes (MAGs) of diverse bacteria and archaea encoded a remarkable diversity of metabolic pathways, including those associated with carbon fixation, carbon monoxide oxidation, acetogenesis, methanogenesis, sulfide oxidation, denitrification, and nitrogen fixation. These results demonstrate the potential for multiple energy sources and metabolic pathways in BSF and highlight the possibility for vibrant microbial ecosystems in the shallow subsurface of evaporites.

Functional Metabolic Diversity of Bacterioplankton in Maritime Antarctic Lakes

A summer survey was conducted on the bacterioplankton communities of seven lakes from Byers Peninsula (Maritime Antarctica), differing in trophic and morphological characteristics. Predictions of the metabolic capabilities of these communities were performed with FAPROTAX using 16S rRNA sequencing data. The versatility for metabolizing carbon sources was also assessed in three of the lakes using Biolog Ecoplates. Relevant differences among lakes and within lake depths were observed. A total of 23 metabolic activities associated to the main biogeochemical cycles were foreseen, namely, carbon (11), nitrogen (4), sulfur (5), iron (2), and hydrogen (1). The aerobic metabolisms dominated, although anaerobic respiration was also relevant near the lakes’ bottom as well as in shallow eutrophic lakes with higher nutrient and organic matter contents. Capacity for using carbon sources further than those derived from the fresh autochthonous primary production was detected. Clustering of the lakes based on metabolic capabilities of their microbial communities was determined by their trophic status, with functional diversity increasing with trophic status. Data were also examined using a co-occurrence network approach, indicating that the lakes and their catchments have to be perceived as connected and interacting macrosystems, where either stochastic or deterministic mechanisms for the assembling of communities may occur depending on the lake’s isolation. The hydrological processes within catchments and the potential metabolic plasticity of these biological communities must be considered for future climate scenarios in the region, which may extend the growing season and increase biomass circulation.

Extracellular Electron Transfer Enables Cellular Control of Cu(I)-catalyzed Alkyne-Azide Cycloaddition

Extracellular electron transfer (EET) is an anaerobic respiration process that couples carbon oxidation to the reduction of metal species. In the presence of a suitable metal catalyst, EET allows for cellular metabolism to control a variety of synthetic transformations. Here, we report the use of EET from the model electroactive bacterium Shewanella oneidensis for metabolic and genetic control over Cu(I)-catalyzed Alkyne-Azide Cycloaddition (CuAAC). CuAAC conversion under anaerobic and aerobic conditions was dependent on live, actively respiring S. oneidensis cells. In addition, reaction progress and kinetics could be further manipulated by tailoring the central carbon metabolism of S. oneidensis. Similarly, CuAAC activity was dependent on specific EET pathways and could be manipulated using inducible genetic circuits controlling the expression of EET-relevant proteins including MtrC, MtrA, and CymA. EET-driven CuAAC also exhibited modularity and robustness in ligand tolerance and substrate scope. Furthermore, the living nature of this system could be exploited to perform multiple reaction cycles without requiring regeneration, something inaccessible to traditional chemical reductants. Finally, S. oneidensis enabled bioorthogonal CuAAC membrane labelling on live mammalian cells without affecting cell viability, suggesting that S. oneidensis can act as a dynamically tunable biocatalyst in complex environments. In summary, our results demonstrate how EET can expand the reaction scope available to living systems by enabling cellular control of CuAAC.