Identification of bacterial dissimilatory antimonate reductase AnrA: genes and proteins involved in antimonate respiration and resistance in Geobacter sp. strain SVR.

Geobacter sp. strain SVR uses antimonate [Sb(V)] as a terminal electron acceptor for anaerobic respiration. Here, we visualized a possible key enzyme, periplasmic Sb(V) reductase (Anr), via active staining and non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that a novel dimethyl sulfoxide (DMSO) reductase family protein, WP_173201954.1, is involved in Anr. This protein was closely related with AnrA, a protein suggested to be the catalytic subunit of a respiratory Sb(V) reductase in Desulfuribacillus stibiiarsenatis. The anr genes of strain SVR (anrXSRBAD) formed an operon-like structure, and their transcription was upregulated under Sb(V)-respiring conditions. The expression of anrA gene was induced by more than 1 µM of antimonite [Sb(III)]; however, arsenite [As(III)] did not induce the expression of anrA gene. Tandem mass tag-based proteomic analysis revealed that, in addition to Anr proteins, proteins in the following categories were upregulated under Sb(V)-respiring conditions: (i) Sb(III) efflux systems such as Ant and Ars; (ii) antioxidizing proteins such as ferritin, rubredoxin, and thioredoxin; (iii) protein quality control systems such as HspA, HslO, and DnaK; and (iv) DNA repair proteins such as UspA and UvrB. These results suggest that strain SVR copes with antimony stress by modulating pleiotropic processes to resist and actively metabolize antimony. To the best of our knowledge, this is the first report to demonstrate the involvement of AnrA in Sb(V) respiration at the protein level. Furthermore, this is the first example to show high expression of the Ant system proteins in the Sb(V)-respiring bacterium.IMPORTANCEAntimony (Sb) exists mainly as antimonite [Sb(III)] or antimonate [Sb(V)] in the environment, and Sb(III) is more toxic than Sb(V). Recently, microbial involvement in Sb redox reactions has received attention. Although more than 90 Sb(III)-oxidizing bacteria have been reported, information on Sb(V)-reducing bacteria is limited. Especially, the enzyme involved in dissimilatory Sb(V) reduction, or Sb(V) respiration, is unclear, despite this pathway being very important for the circulation of Sb in nature. In this study, we demonstrated that the Sb(V) reductase (Anr) of an Sb(V)-respiring bacterium (Geobacter sp. SVR) is a novel member of the dimethyl sulfoxide (DMSO) reductase family. In addition, we found that strain SVR copes with Sb stress by modulating pleiotropic processes, including the Ant and Ars systems, and upregulating the antioxidant and quality control protein levels. Considering the abundance and diversity of putative anr genes in the environment, Anr may play a significant role in global Sb cycling in both marine and terrestrial environments.

Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Pseudomonas aeruginosa Activates Quorum Sensing, Antioxidant Enzymes and Type VI Secretion in Response to Oxidative Stress to Initiate Biofilm Formation and Wound Chronicity.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen frequently isolated from cutaneous chronic wounds. How PA, in the presence of oxidative stress (OS), colonizes chronic wounds and forms a biofilm is still unknown. The purpose of this study is to investigate the changes in gene expression seen when PA is challenged with the high levels of OS present in chronic wounds. We used a biofilm-forming PA strain isolated from the chronic wounds of our murine model (RPA) and performed a qPCR to obtain gene expression patterns as RPA developed a biofilm in vitro in the presence of high levels of OS, and then compared the findings in vivo, in our mouse model of chronic wounds. We found that the planktonic bacteria under OS conditions overexpressed quorum sensing genes that are important for the bacteria to communicate with each other, antioxidant stress genes important to reduce OS in the microenvironment for survival, biofilm formation genes and virulence genes. Additionally, we performed RNAseq in vivo and identified the activation of novel genes/pathways of the Type VI Secretion System (T6SS) involved in RPA pathogenicity. In conclusion, RPA appears to survive the high OS microenvironment in chronic wounds and colonizes these wounds by turning on virulence, biofilm-forming and survival genes. These findings reveal pathways that may be promising targets for new therapies aimed at disrupting PA-containing biofilms immediately after debridement to facilitate the treatment of chronic human wounds.

Demystifying anaerobic respiration: a problem-solving exercise.

Anaerobic respiration reactions are of fundamental importance to global biogeochemical cycling of elements. Yet, the idea that cellular respiration can occur not only in the absence of oxygen but also involve the oxidation of inorganic substrates (e.g., AsO33-, Fe2+, H2, H2S, Mn2+, NH3, and S0) is often foreign to many undergraduate students. This article describes a problem-solving exercise where students are introduced to the thermodynamic fundamentals of respiration with a particular focus on the role of redox (reduction-oxidation) potentials (E0´). In the exercise, the students investigate how the difference in redox potential (ΔE0´) between different pairs of reductants and oxidants affects the range of permissible microbial metabolic reactions in natural environments when oxygen is absent.

Iron limitation indirectly reduces the Escherichia coli torCAD operon expression by a reduction of molybdenum cofactor availability.

The expression of most molybdoenzymes in Escherichia coli has so far been revealed to be regulated by anaerobiosis and requires the presence of iron, based on the necessity of the transcription factor FNR to bind one [4Fe-4S] cluster. One exception is trimethylamine-N-oxide reductase encoded by the torCAD operon, which has been described to be expressed independently from FNR. In contrast to other alternative anaerobic respiratory systems, the expression of the torCAD operon was shown not to be completely repressed by the presence of dioxygen. To date, the basis for the O2-dependent expression of the torCAD operon has been related to the abundance of the transcriptional regulator IscR, which represses the transcription of torS and torT, and is more abundant under aerobic conditions than under anaerobic conditions. In this study, we reinvestigated the regulation of the torCAD operon and its dependence on the presence of iron and identified a novel regulation that depends on the presence of the bis-molybdopterin guanine dinucleotide (bis-MGD) molybdenum cofactor . We confirmed that the torCAD operon is directly regulated by the heme-containing protein TorC and is indirectly regulated by ArcA and by the availability of iron via active FNR and Fur, both regulatory proteins that influence the synthesis of the molybdenum cofactor. Furthermore, we identified a novel regulation mode of torCAD expression that is dependent on cellular levels of bis-MGD and is not used by other bis-MGD-containing enzymes like nitrate reductase.IMPORTANCEIn bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. FNR is a very important transcription factor that represents the master switch for the expression of target genes in response to anaerobiosis. Only Escherichia coli trimethylamine-N-oxide (TMAO) reductase escapes this regulation by FNR. We identified that the expression of TMAO reductase is regulated by the amount of bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor synthesized by the cell itself, representing a novel regulation pathway for the expression of an operon coding for a molybdoenzyme. Furthermore, TMAO reductase gene expression is indirectly regulated by the presence of iron, which is required for the production of the bis-MGD cofactor in the cell.

The resting cyst of dinoflagellate Scrippsiella acuminata host bacterial microbiomes with more diverse trophic strategies under conditions typically observed in marine sediments.

Variation in the condition of marine sediments provides selective preservation milieus, which act as a key determinant for the abundance and distribution of dinoflagellate resting cysts in natural sediments. Microbial degradation is an understudied biological factor of potential importance in the processes. However, gaps remain in our knowledge about the fundamental information of the bacterial consortia associated with dinoflagellate resting cysts both in laboratory cultures and in the field. Here we used Scrippsiella acuminata as a representative of cyst-producing dinoflagellates to delineate the diversity and composition of bacterial microbiomes co-existing with the laboratory-cultured resting cysts, and to explore possible impacts of low temperature, darkness, and anoxia (the mock conditions commonly observed in marine sediments) on the associated bacterial consortia. Bacterial microbiome with high diversity were revealed associated with S. acuminata at resting stage. The mock conditions could significantly shift bacterial community structure and exert notably inhibitory effects on growth-promoting bacteria. Resting cysts under conditions typically observed in marine sediments fostered bacterial microbiomes with more diverse trophic strategies, characteristic of prominently enriched anaerobic chemotrophic bacteria generating energy via respiration with several different terminal electron acceptors, which yielded more acidic milieu unfavorable for the preservation of calcareous resting cysts. Our findings suggest that there is complex and dynamic interaction between dinoflagellates resting cysts and the associated bacterial consortia in natural sediments. This intrinsic interaction may influence the maintenance and/or accumulation of dinoflagellate resting cysts with potential of germination and initiation blooms in the field.