Mechanistic insights into tris(2-chloroisopropyl) phosphate biomineralization coupled with lead (II) biostabilization driven by denitrifying bacteria.

The ubiquity and persistence of organophosphate esters (OPEs) and heavy metal (HMs) pose global environmental risks. This study explored tris(2-chloroisopropyl)phosphate (TCPP) biomineralization coupled to lead (Pb2+) biostabilization driven by denitrifying bacteria (DNB). The domesticated DNB achieved synergistic bioremoval of TCPP and Pb2+ in the batch bioreactor (efficiency: 98 %).TCPP mineralized into PO43- and Cl-, and Pb2+ precipitated with PO43-. The TCPP-degrading/Pb2+-resistant DNB: Achromobacter, Pseudomonas, Citrobacter, and Stenotrophomonas, dominated the bacterial community, and synergized TCPP biomineralization and Pb2+ biostabilization. Metagenomics and metaproteomics revealed TCPP underwent dechlorination, hydrolysis, the TCA cycle-based dissimilation, and assimilation; Pb2+ was detoxified via bioprecipitation, bacterial membrane biosorption, EPS biocomplexation, and efflux out of cells. TCPP, as an initial donor, along with NO3-, as the terminal acceptor, formed a respiratory redox as the primary energy metabolism. Both TCPP and Pb2+ can stimulate phosphatase expression, which established the mutual enhancements between their bioconversions by catalyzing TCPP dephosphorylation and facilitating Pb2+ bioprecipitation. TCPP may alleviate the Pb2+-induced oxidative stress by aiding protein phosphorylation. 80 % of Pb2+ converted into crystalized pyromorphite. These results provide the mechanistic foundations and help develop greener strategies for synergistic bioremediation of OPEs and HMs.

Three-component systems represent a common pathway for extracytoplasmic addition of pentofuranose sugars into bacterial glycans.

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.

Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

Pangenome-driven insights into nitrogen metabolic characteristics of Citrobacter portucalensis strain AAK_AS5 associated with wastewater nitrogen removal.

Nitrogen metabolism in the genus Citrobacter is very poorly studied despite its several implications in wastewater treatment. In the current study, Citrobacter portucalensis strain AAK_AS5 was assessed for remediation of simulated wastewater supplemented with different inorganic nitrogen sources. Combination of (NH4)2SO4 with KNO3 was the most preferred for achieving high growth density followed by (NH4)2SO4 and KNO3 alone. This was in agreement with highest ammonical nitrogen removal of 92.9% in the presence of combined nitrogen sources and the corresponding nitrate nitrogen removal of 93% in the presence of KNO3. Furthermore, these removal capacities were validated by investigating the uniqueness and the spread of metabolic features through pan-genomic approach that revealed the largest number of unique genes (2097) and accessory genes (705) in strain AAK_AS5. Of the total 44 different types of nitrogen metabolism-related genes, 39 genes were associated with the core genome, while 5 genes such as gltI, nasA, nasR, nrtA, and ntrC uniquely belonged to the accessory genome. Strain AAK_AS5 possessed three major nitrate removal pathways viz., assimilatory and dissimilatory nitrate reduction to ammonia (ANRA & DNRA), and denitrification; however, the absence of nitrification was compensated by ammonia assimilation catalyzed by gene products of the GDH and GS-GOGAT pathways. narGHIJ encoding the respiratory nitrate reductase was commonly identified in all the studied genomes, while genes such as nirK, norB, and nosZ were uniquely present in the strain AAK_AS5 only. A markedly different genetic content and metabolic diversity between the strains reflected their adaptive evolution in the environment thus highlighting the significance of C. portucalensis AAK_AS5 for potential application in nitrogen removal from wastewater.

The trafficking of HgII by alleviating its toxicity via Citrobacter sp. IITISM25 in batch and pilot-scale investigation.

The study aims to see how effective the Citrobacter species strain is in removing HgII under stressful conditions. For this, a response surface methodology was chosen to optimized pH, temperature, and biomass for effective biotransformation of HgII. The optimized value for pH, temperature, and biomass were 6.5, 30 °C, and 2 mg/l with 89% HgII removal potential. TEM-EDX showed accumulated mercury onto the bacterial surface. Pot study was conducted to check the potentiality of this strain in alleviating the toxicity in Solanum lycopersicum L. under different concentrations of mercury. The enhancement in antioxidative enzymes, as well as mercury accumulation, was observed in test plants inoculated with IITISM25. Obtained result showed a greater accumulation of mercury in the root system than that of the shoot system due to poor translocation. Moreover, mercury reductase enzyme synthesis was also boosted by the addition of ß-mercaptoethanol and L-cysteine. The optimized condition for maximum enzyme synthesis was at pH 7.5 and temperature 30 °C with Km = 48.07 µmol and Vmax = 9.75 µmol/min. Thus, we can say that Citrobacter species strain IITISM25 can be effectively applied in remediation of HgII stress condition as well as promotion of Solanum lycopersicum L growth under stress conditions as a promising host.

Molecular Cloning, Expression and Characterisation of a Bacterial Myrosinase from Citrobacter Wye1.

Glucosinolates are plant natural products which on degradation by myrosinases give rise to the beneficial bioactive isothiocyanates. Recently, a myrosinase activity was detected in a Citrobacter strain isolated from soil. This enzyme was purified enabling its amino acid sequence and gene sequence (cmyr) to be determined. In order to study this myrosinase it was necessary to establish an expression system that would enable future work such as a structural determination of the protein to be carried out. The myrosinase gene was amplified, cloned and expressed in Escherichia coli with a 6XHis-tag. The heterologous expression of cmyr enabled relatively large amounts of myrosinase to be produced (3.4 mg cmyr/100 ml culture). Myrosinase activity was determined by mixing substrate and enzyme and determining glucose release. Optimum pH and temperature were determined to be pH 6.0 and 25 °C for the Ni-NTA purified protein. The kinetic parameters of the purified myrosinase were determined using sinigrin as a substrate. Km and Vmax were estimated as 0.18 mM and 0.033 mmol/min/mg respectively for sinigrin under optimum conditions and compared to other kinetic data for myrosinases. The substrate specificity of myrosinase was determined having the highest affinity for sinigrin followed by glucoiberin, progoitrin, glucoerucin, glucoraphanin and glucotropaeolin.

Nitrate Reduction Stimulates and Is Stimulated by Phenazine-1-Carboxylic Acid Oxidation by Citrobacter portucalensis MBL.

Phenazines are secreted metabolites that microbes use in diverse ways, from quorum sensing to antimicrobial warfare to energy conservation. Phenazines are able to contribute to these activities due to their redox activity. The physiological consequences of cellular phenazine reduction have been extensively studied, but the counterpart phenazine oxidation has been largely overlooked. Phenazine-1-carboxylic acid (PCA) is common in the environment and readily reduced by its producers. Here, we describe its anaerobic oxidation by Citrobacter portucalensis strain MBL, which was isolated from topsoil in Falmouth, MA, and which does not produce phenazines itself. This activity depends on the availability of a suitable terminal electron acceptor, specifically nitrate. When C. portucalensis MBL is provided reduced PCA and nitrate, it oxidizes the PCA at a rate that is environmentally relevant. We compared this terminal electron acceptor-dependent PCA-oxidizing activity of C. portucalensis MBL to that of several other gammaproteobacteria with various capacities to respire nitrate. We found that PCA oxidation by these strains in a nitrate-dependent manner is decoupled from growth and strain dependent. We infer that bacterial PCA oxidation is widespread and genetically determined. Notably, oxidizing PCA enhances the rate of nitrate reduction to nitrite by C. portucalensis MBL beyond the stoichiometric exchange of electrons from PCA to nitrate, which we attribute to C. portucalensis MBL's ability to also reduce oxidized PCA, thereby catalyzing a complete PCA redox cycle. This bidirectionality highlights the versatility of PCA as a biological redox agent. IMPORTANCE Phenazines are increasingly appreciated for their roles in structuring microbial communities. These tricyclic aromatic molecules have been found to regulate gene expression, be toxic, promote antibiotic tolerance, and promote survival under oxygen starvation. In all of these contexts, however, phenazines are studied as electron acceptors. Even if their utility arises primarily from being readily reduced, they need to be oxidized in order to be recycled. While oxygen and ferric iron can oxidize phenazines abiotically, biotic oxidation of phenazines has not been studied previously. We observed bacteria that readily oxidize phenazine-1-carboxylic acid (PCA) in a nitrate-dependent fashion, concomitantly increasing the rate of nitrate reduction to nitrite. Because nitrate is a prevalent terminal electron acceptor in diverse anoxic environments, including soils, and phenazine producers are widespread, this observation of linked phenazine and nitrogen redox cycling suggests an underappreciated role for redox-active secreted metabolites in the environment.

Insights into evolution and coexistence of the colibactin- and yersiniabactin secondary metabolite determinants in enterobacterial populations.

The bacterial genotoxin colibactin interferes with the eukaryotic cell cycle by causing dsDNA breaks. It has been linked to bacterially induced colorectal cancer in humans. Colibactin is encoded by a 54 kb genomic region in Enterobacteriaceae. The colibactin genes commonly co-occur with the yersiniabactin biosynthetic determinant. Investigating the prevalence and sequence diversity of the colibactin determinant and its linkage to the yersiniabactin operon in prokaryotic genomes, we discovered mainly species-specific lineages of the colibactin determinant and classified three main structural settings of the colibactin-yersiniabactin genomic region in Enterobacteriaceae. The colibactin gene cluster has a similar but not identical evolutionary track to that of the yersiniabactin operon. Both determinants could have been acquired on several occasions and/or exchanged independently between enterobacteria by horizontal gene transfer. Integrative and conjugative elements play(ed) a central role in the evolution and structural diversity of the colibactin-yersiniabactin genomic region. Addition of an activating and regulating module (clbAR) to the biosynthesis and transport module (clbB-S) represents the most recent step in the evolution of the colibactin determinant. In a first attempt to correlate colibactin expression with individual lineages of colibactin determinants and different bacterial genetic backgrounds, we compared colibactin expression of selected enterobacterial isolates in vitro. Colibactin production in the tested Klebsiella species and Citrobacter koseri strains was more homogeneous and generally higher than that in most of the Escherichia coli isolates studied. Our results improve the understanding of the diversity of colibactin determinants and its expression level, and may contribute to risk assessment of colibactin-producing enterobacteria.

Effect of two phytases at two doses on performance and phytate degradation in broilers during 1-21 days of age.

The effect of two microbial phytases at two dose-levels on performance and apparent ileal digestibility (AID) of nutrients in broilers fed European-type diets was studied. A total of 1,200 d-old Ross 308 male broilers were randomly assigned to 5 treatments with 30 birds/pen and 8 pens/treatment. A nutritionally adequate positive control (PC) diet was tested against 4 experimental diets containing reduced total P, retainable P, Ca and Na as per the recommended nutritional contribution for Buttiauxella phytase (Phy B) at 1,000 FTU/kg (-1.87 g/kg, -1.59 g/kg, -1.99 g/kg and -0.4 g/kg vs. PC, respectively). Experimental diets were supplemented with Phy B at 500 FTU/kg or 1,000 FTU/kg, or Citrobacter phytase (Phy C) at 1,000 FTU/kg or 2,000 FTU/kg. Diets were based on corn, soybean meal, rapeseed meal and sunflower meal and formulated by phase (starter 1-10 d, grower 11-21 d) in crumbled or pelleted form. Overall (d 1-21), at 1,000 FTU/kg, birds fed Phy C exhibited lower BWG (-2.7%), FI (-3.4%) and tibia ash (-2.2%) vs. PC (P < 0.05), and reduced BWG (-3.6%), FI (-3.9%) and tibia ash (-1.8%) vs. Phy B (P < 0.05). Phy B at 1,000 FTU/kg and Phy C at 2,000 FTU/kg maintained performance equivalent to the PC. Digestibility of Ca did not differ among phytase treatments but at 1,000 FTU/kg AID P was greater with Phy B than Phy C (72.3% vs. 62.7%, P < 0.05). Ileal phytate (myo-inositol hexakisphosphate, IP6) digestibility was greatest with Phy B at 1,000 FTU/kg which was higher than Phy C at 1,000 FTU/kg (87.6 vs. 60.6%, P < 0.05). The findings indicate a higher phytate degradation rate of Phy B than Phy C at equivalent dose-level and this is correlated to the performance of the broilers.

Insights on intermolecular FMN-heme domain interaction and the role of linker length in cytochrome P450cin fusion proteins.

Cytochrome P450s are an important group of enzymes catalyzing hydroxylation, and epoxidations reactions. In this work we describe the characterization of the CinA-CinC fusion enzyme system of a previously reported P450 using genetically fused heme (CinA) and FMN (CinC) enzyme domains from Citrobacter braaki. We observed that mixing individually inactivated heme (-) with FMN (-) domain in the CinA-10aa linker - CinC fusion constructs results in recovered activity and the formation of (2S)-2ß-hydroxy,1,8-cineole (174 µM), a similar amount when compared to the fully functional fusion protein (176 µM). We also studied the effect of the fusion linker length in the activity complementation assay. Our results suggests an intermolecular interaction between heme and FMN parts from different CinA-CinC fusion protein similar to proposed mechanisms for P450 BM3 on the other hand, linker length plays a crucial influence on the activity of the fusion constructs. However, complementation assays show that inactive constructs with shorter linker lengths have functional subunits, and that the lack of activity might be due to incorrect interaction between fused enzymes.

Anti-Bacterial and Anti-Candidal Activity of Silver Nanoparticles Biosynthesized Using Citrobacter spp. MS5 Culture Supernatant.

The present study described the extracellular synthesis of silver nanoparticles (AgNPs) using environmental bacterial isolate Citrobacter spp. MS5 culture supernatant. To our best knowledge, no previous study reported the biosynthesis of AgNPs using this bacterial isolate. The biosynthesized AgNPs were characterized using different techniques like UV-Vis spectroscopy, fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) equipped with energy dispersive X-ray (EDX). The analysis of UV-Vis spectra revealed absorption maxima at 415 nm due to surface plasmon resonance (SPR) indicated the formation of AgNPs and FTIR spectrum confirmed the participation of proteins molecule in AgNPs synthesis. XRD and EDX spectrum confirmed the metallic and crystalline nature of AgNPs. TEM and SEM showed spherical nanoparticles with a size range of 5-15 nm. The biosynthesized AgNPs showed effective independent as well as enhanced combined antibacterial activity against extended spectrum ß-lactamase (ESBL) producing multidrug resistant Gram-negative bacteria. Further, effective antifungal activity of AgNPs was observed towards pathogenic Candida spp. The present study provides evidence for eco-friendly biosynthesis of well-characterized AgNPs and their potential antibacterial as well as antifungal activity.

Mutations in the two component regulator systems PmrAB and PhoPQ give rise to increased colistin resistance in Citrobacter and Enterobacter spp.

Introduction. Colistin is a last resort antibiotic for treating infections caused by carbapenem-resistant isolates. Mechanisms of resistance to colistin have been widely described in Klebsiella pneumoniae and Escherichia coli but have yet to be characterized in Citrobacter and Enterobacter species.Aim. To identify the causative mutations leading to generation of colistin resistance in Citrobacter and Enterobacter spp.Methodology. Colistin resistance was generated by culturing in increasing concentrations of colistin or by direct culture in a lethal (above MIC) concentration. Whole-genome sequencing was used to identify mutations. Fitness of resistant strains was determined by changes in growth rate, and virulence in Galleria mellonella.Results. We were able to generate colistin resistance upon exposure to sub-MIC levels of colistin, in several but not all strains of Citrobacter and Enterobacter resulting in a 16-fold increase in colistin MIC values for both species. The same individual strains also developed resistance to colistin after a single exposure at 10× MIC, with a similar increase in MIC. Genetic analysis revealed that this increased resistance was attributed to mutations in PmrB for Citrobacter and PhoP in Enterobacter, although we were not able to identify causative mutations in all strains. Colistin-resistant mutants showed little difference in growth rate, and virulence in G. mellonella, although there were strain-to-strain differences.Conclusions. Stable colistin resistance may be acquired with no loss of fitness in these species. However, only select strains were able to adapt suggesting that acquisition of colistin resistance is dependent upon individual strain characteristics.

Screening of tea saponin-degrading strain to degrade the residual tea saponin in tea seed cake.

Although tea seed cake (TSC) possesses high nutritional value, its high content of tea saponin (TS) limits its potential as feed. This study aimed to degrade TS in TSC by saponin-degrading strain and used a multistrains fermentation method to improve its nutritional value and palatability. Three saponin-degrading strains were isolated from Oleum Camelliae mill soil and identified as Citrobacter sp. FCTS301, Pantoea sp. FCTS302, and Enterobacter sp. FCTS303. Single-factor experiment showed that Citrobacter sp. FCTS301 had the highest degradation rate of TS. Response surface analysis for Citrobacter sp. FCTS301 indicated that the optimum culture conditions were as follows: initial pH of 7.2, culture temperature of 34.2 °C, inoculation amount of 7.3%, the agitation rate of 150 rpm, and the TS concentration of 10.0 g/L. Under these conditions, the maximum degradation rate was 82.6%. The fermentation process of TSC was obtained by a multistrains fermentation experiment. Considering the protein content, crude fiber degradation rate, and TS degradation rate of each group, the optimum inoculum amount of strains included Citrobacter sp. FCTS301, Aspergillus oryzae NCUF414, Saccharomyces cersvisiae NCUF306.5, and Lactobacillus plantarum NCUF201.1(5%, 0.5%, 1.0%, and 1.5%). After TS was degraded efficiently, fermented TSC can be presumed a potential feed raw material.

Crystal structures of AztD provide mechanistic insights into direct zinc transfer between proteins.

Zinc acquisition from limited environments is critical for bacterial survival and pathogenesis. AztD has been identified as a periplasmic or cell surface zinc-binding protein in numerous bacterial species. In Paracoccus denitrificans, AztD can transfer zinc directly to AztC, the solute binding protein for a zinc-specific ATP-binding cassette transporter system, suggesting a role in zinc acquisition and homeostasis. Here, we present the first cry stal structures of AztD from P. denitrificans and tbe human pathogen Citrobacter koseri, revealing a beta-propeller fold and two high-affinity zinc-binding sites that are highly conserved among AztD homologs. These structures combined with transfer assays using WT and mutant proteins provide rare insight into the mechanism of direct zinc transfer from one protein to another. Given the importance of zinc import to bacterial pathogenesis, these insights may prove valuable to the development of zinc transfer inhibitors as antibiotics.

Unprecedented Microbial Conversion of Biliverdin into Bilirubin-10-sulfonate.

Biliverdin (BV) possesses antioxidant and anti-inflammatory properties, with previous reports identifying protection against oxidant and inflammatory injury in animal models. Recent reports indicate that intra-duodenal administration of BV results in the formation of an uncharacterised metabolite, which is potently absorbed into the blood and excreted into the bile. This compound may be responsible for protection against inflammatory responses. This study aimed to identify novel, enterally-derived BV metabolites and determine the source of their metabolic transformation. Rat duodena and bacterial cultures of Citrobacter youngae were treated with BV and subsequently analysed via high performance liquid chromatography/high resolution tandem mass spectrometry to identify and characterise metabolites of BV. A highly abundant metabolite was detected in duodenal wash and bacterial culture supernatants with a 663.215 m/z (3 ppm mass accuracy) and a composition of C33N4O9H36S, which conformed to the predicted structure of bilirubin-10-sulfonate (BRS) and possessed a λmax of 440 nm. Bilirubin-10-sulfonate was then synthesized for comparative LCMS/MS analysis and matched with that of the biologically formed BV metabolite. This report confirms the formation of a previously undocumented metabolite of BV in mammals, indicating that a new metabolic pathway likely exists for BV metabolism requiring enteric bacteria, Citrobacter youngae. These data may have important implications with regard to understanding and harnessing the therapeutic efficacy of oral BV administration.

Characterization of quorum sensing genes and N-acyl homoserine lactones in Citrobacter amalonaticus strain YG6.

In the phylum of Proteobacteria, quorum sensing (QS) system is widely driven by synthesis and response of N-acyl homoserine lactone (AHL) signalling molecules. AHL is synthesized by LuxI homologue and sensed by LuxR homologue. Once the AHL concentration achieves a threshold level, it triggers the regulation of target genes. In this study, QS activity of Citrobacter amalonaticus strain YG6 which was isolated from clams was investigated. In order to characterise luxI/R homologues, the genome of C. amalonaticus strain YG6 (4.95 Mbp in size) was sequenced using Illumina MiSeq sequencer. Through in silico analysis, a pair of canonical luxI/R homologues and an orphan luxR homologue were identified and designated as camI, camR, and camR2, respectively. A putative lux box was identified at the upstream of camI. The camI gene was cloned and overexpressed in E. coli BL21 (DE3)pLysS. High-resolution triple quadrupole liquid chromatography mass spectrometry (LC-MS/MS) analysis verified that the CamI is a functional AHL synthase which produced multiple AHL species, namely N­butyryl­l­homoserine lactone (C4-HSL), N­hexanoyl­l­homoserine lactone (C6-HSL), N­octanoyl­l­homoserine lactone (C8-HSL), N­tetradecanoyl­l­homoserine lactone (C14-HSL) and N­hexadecanoyl­l­homoserine lactone (C16-HSL) in C. amalonaticus strain YG6 and camI gene in recombinant E. coli BL21(DE3)pLysS. To our best knowledge, this is the first functional study report of camI as well as the first report describing the production of C14-HSL by C. amalonaticus.

Arsenate-dependent growth is independent of an ArrA mechanism of arsenate respiration in the termite hindgut isolate Citrobacter sp. strain TSA-1.

Citrobacter sp. strain TSA-1 is an enteric bacterium isolated from the hindgut of the termite. Strain TSA-1 displays anaerobic growth with selenite, fumarate, tetrathionate, nitrate, or arsenate serving as electron acceptors, and it also grows aerobically. In regards to arsenate, genome sequencing revealed that strain TSA-1 lacks a homolog for respiratory arsenate reductase, arrAB, and we were unable to obtain amplicons of arrA. This raises the question as to how strain TSA-1 achieves As(V)-dependent growth. We show that growth of strain TSA-1 on glycerol, which it cannot ferment, is linked to the electron acceptor arsenate. A series of transcriptomic experiments were conducted to discern which genes were upregulated during growth on arsenate, as opposed to those on fumarate or oxygen. For As(V), upregulation was noted for 1 of the 2 annotated arsC genes, while there was no clear upregulation for tetrathionate reductase (ttr), suggesting that this enzyme is not an alternative to arrAB as occurs in certain hyperthermophilic archaea. A gene-deletion mutant strain of TSA-1 deficient in arsC could not achieve anaerobic respiratory growth on As(V). Our results suggest that Citrobacter sp. strain TSA-1 has an unusual and as yet undefined means of achieving arsenate respiration, perhaps involving its ArsC as a respiratory reductase as well as a detoxifying agent.

Electrode plate-culture methods for colony isolation of exoelectrogens from anode microbiomes.

Exoelectrogens play central roles in microbial fuel cells and other bioelectrochemical systems (BESs), yet their physiological diversity remains largely elusive due to the lack of efficient methods for the isolation from naturally occurring microbiomes. The present study developed an electrode plate-culture (EPC) method that facilitates selective colony formation by exoelectrogens and used it for isolating them from an exoelectrogenic microbiome enriched from paddy-field soil. In an EPC device, the surface of solidified agarose medium was spread with a suspension of a microbiome and covered with a transparent fluorine doped tin oxide (FTO) electrode (poised at 0 V vs. the standard hydrogen electrode) that served as the sole electron acceptor. The medium contained acetate as the major growth substrate and Coomassie Brilliant Blue as a dye for visualizing colonies under FTO. It was shown that colonies successfully appeared under FTO in association with current generation. Analyses of 16S rRNA gene sequences of colonies indicated that they were affiliated with genera Citrobacter, Geobacter and others. Among them, Citrobacter and Geobacter isolates were found to be exoelectrogenic in pure-culture BESs. These results demonstrate the utility of the EPC method for colony isolation of exoelectrogens.

The integrated analysis of transcriptome and proteome for exploring the biodegradation mechanism of 2, 4, 6-trinitrotoluene by Citrobacter sp.

Citrobacter sp. has been shown to degrade 2,4,6-trinitrotoluene (TNT). However, the mechanism of its TNT biodegradation is poorly understood. An integrated proteome and transcriptome analysis was performed for investigating the differential genes and differential proteins in bacterial growth at the onset of experiments and after 12 h treatment with TNT. With the RNA sequencing, we found a total of 3792 transcripts and 569 differentially expressed genes (≥2 fold, P < 0.05) by. Genes for amino acid transport, cellular metabolism and stress-shock proteins were up-regulated, while carbohydrate transport and metabolism were down-regulated. A total of 42 protein spots (≥1.5 fold, P < 0.05) showed differential expression on two-dimensional gel electrophoresis and these proteins were identified by mass spectrometry. The most prominent proteins up-regulated were involved in energy production and conversion, amino acid transport and metabolism, posttranslational modification, protein turnover and chaperones. Proteins involved in carbohydrate transport and metabolism were down-regulated. Most notably, we observed that nemA encoding N-ethylmaleimide reductase was the most up-regulated gene involved in TNT degradation, and further proved that it can transform TNT to 4-amino-2,6-dinitrotoluene (4-ADNT) and 2-amino-4,6-dinitrotoluene (2-ADNT). This study highlights the molecular mechanisms of Citrobacter sp. for TNT removal.

Phenol biodegradation by isolated Citrobacter strain under hypersaline conditions.

Phenol is a toxic pollutant in many kinds of hypersaline industrial effluents that should be treated properly before discharged into water bodies. In this work, a halophilic strain which could utilize phenol as the sole source of carbon and energy was isolated. Based on 16S rRNA results, it was identified as a member of Citrobacter. The phenol biodegradation ability and cell growth of the strain was evaluated with the variation of initial phenol concentration and salinity. The effect of temperature and pH on phenol removal was also investigated. The results showed that the strain was capable of withstanding high phenol (up to 1,100 mg L-1) environment with varying salinity conditions (0-10% of NaCl). The optimal initial phenol concentration was 400 mg L-1, at which the average removal rates of phenol peaked at 10.8 mg L-1 h-1. The higher initial concentration of phenol could inhibit the microbial metabolism. The optimal temperature, pH, and salinity were 35 °C, 6.0, and 0%, respectively. Under these conditions, 400 mg L-1 of phenol could be completely degraded within 20 h. The high removal rates of phenol by the strain might provide an alternative for treating phenolic wastewaters containing high salinity.