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Ammonium sulfate ((NH 4 ) 2 SO 4 ) leaching method to extract rare earth elements (REEs) of mine has produced a large amount of NH 4 + -N-enriched wastewater derived from ore body, leading to many serious environmental pollution problems. This study was the first time to establish an in-situ treatment for real REEs wastewater outside and inside the ore body by an isolated indigenous microorganism. The results stated that Citrobacter sp. X-9 achieved the highest NH 4 + -N removal efficiency among the isolated six microbial strains. Moreover, the microbe to treat the REEs wastewater outside ore body gave the greatest NH 4 + -N removal efficiency under the optimized conditions in the Erlenmeyer flask (250-mL) and bioreactor (10-L). Furthermore, compared to the others' modes, the in-situ treatment by cyclic mode with Citrobacter sp. X-9 possessed superior performance in NH 4 + -N removal efficiency for wastewater inside of ore body, showing that the established in-situ treatment was the potential approach for REEs wastewater purification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Microcystin-LR (MC-LR), produced by cyanobacterial harmful algal blooms, poses a serious threat to aquatic ecosystems and human health. Biodegradation is an important method for MC-LR elimination. Aerobic biodegradation has been extensively studied and many bacteria were identified. However, few MC-degrading bacteria have been isolated from anaerobic environments, and these degradation mechanisms are poorly understood. The aim of this study was to collect anaerobic MC-degrading bacteria from lake sediments in Lake Taihu using acclimation culture to assess biodegradation. Five strains with MC-LR degradation ability were isolated, with strain A4 belonging to Citrobacter farmeri ( C. farmeri ). exhibiting the highest efficiency at 0.486 μg/ml/d. High-performance liquid chromatography (HPLC) identified two novel MC-LR degradation products. Further polymerase chain reaction (PCR) analysis suggested that C. farmeri A4 did not possess the known MC-degrading gene mlrABCD , suggesting the involvement of an mlrABCD -independent anaerobic degradation pathway. Data demonstrated that the bacterial strain C. farmeri A4 found in Lake Taihu exhibited high anaerobic MC-LR degradation properties, which indicated that anaerobic biodegradation may constitute an important biological method for MC-LR removal in natural environment.

Arsenic (As) poisoning in aquifers is a serious problem worldwide, especially in the middle-Gangetic Plain (MGP) of India. Microbially-mediated As speciation in such aquifers is governed by the arsenate-reductase enzyme, ArsC, encoded by the arsC gene of As-metabolizing bacteria. In this study, ArsC1 (119 aa) and ArsC2 (141 aa) of a highly resistant strain to arsenic, Citrobacter youngae IITK SM2 (CyIITKSM2), isolated from a mixed-oxic MGP groundwater were biochemically characterized. Coupled-arsenate-reductase assay and IC-ICP-MS analysis confirmed that ArsC2 showed higher As(V) reduction than ArsC1 in the dissolved phase, which was consistent with the prominent structural changes in ArsC2 as identified through circular dichroism spectroscopy. Furthermore, the two ArsCs were able to mobilize arsenic from solid-bound arsenate [As(V)-loaded goethite, AsG] predominantly as As(III). However, the total arsenic released in the presence of ArsC2 was ∼38 % and ∼88 % higher, respectively, as compared to the ArsC1-containing and ArsC-free conditions. A process-based model that considered ArsC-mediated As(V) reduction to As(III) in the dissolved phase, and surface complexation of As(V) and As(III) on goethite, suggested that the extent of arsenate binding with ArsC was not affected by whether As(V) was dissolved or was sorbed. However, the catalytic reduction rate was at least an order of magnitude lower in sorbed As(V) than in dissolved As(V). Mutants of ArsC2 exhibited variable but reduced efficiencies compared to the wild-type ArsC2. This reduction may be attributed to the C-terminal loop observed in the AlphaFold predicted structure of ArsC2, which was absent in ArsC1. This comprehensive biochemical and biophysical analysis of the arsenate reductases in Citrobacter youngae could enhance our understanding of the role these microbes play in arsenic mobilization within MGP aquifers., Competing Interests: Declaration of competing interest The authors declare that no competing financial interests exist, and this work has been carried out in compliance with ethical standards., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Phenol and p-cresol are two common toxic small molecules related to various diseases. Existing reports confirmed that high L -tyrosine in the daily diet can increase the concentration of phenolic compounds in blood and urine. L-tyrosine is a common component of protein-rich foods. Some anaerobic bacteria in the gut can convert non-toxic l-tyrosine into these two toxic phenolic compounds, phenol and p-cresol. Existing methods have been constructed for measuring the concentration of phenolic compound in feces. However, there is still a lack of direct visual evidence to measure the phenolic compounds in the intestine. In this study, we aimed to construct a whole-cell biosensor for phenolic compounds detection based on the dmpR, the regulator from the phenol metabolism cluster. The commensal bacterium Citrobacter amalonaticus PS01 was selected and used as the chassis. Compared with the biosensor based on ECN1917, the biosensor PS01[dmpR] could better implant into the mouse gut through gavage and showed a higher sensitive to phenolic compound. And the concentration of phenolic compounds in the intestines could be observed with the help of in vivo imaging system using PS01[dmpR]. This paper demonstrated endogenous phenol synthesis in the gut and the strategy of using commensal bacteria to construct whole-cell biosensors for detecting small molecule compounds in the intestines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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 (Pb 2+ ) biostabilization driven by denitrifying bacteria (DNB). The domesticated DNB achieved synergistic bioremoval of TCPP and Pb 2+ in the batch bioreactor (efficiency: 98 %).TCPP mineralized into PO 4 3- and Cl - , and Pb 2+ precipitated with PO 4 3- . The TCPP-degrading/Pb 2+ -resistant DNB: Achromobacter, Pseudomonas, Citrobacter, and Stenotrophomonas, dominated the bacterial community, and synergized TCPP biomineralization and Pb 2+ biostabilization. Metagenomics and metaproteomics revealed TCPP underwent dechlorination, hydrolysis, the TCA cycle-based dissimilation, and assimilation; Pb 2+ was detoxified via bioprecipitation, bacterial membrane biosorption, EPS biocomplexation, and efflux out of cells. TCPP, as an initial donor, along with NO 3 - , as the terminal acceptor, formed a respiratory redox as the primary energy metabolism. Both TCPP and Pb 2+ can stimulate phosphatase expression, which established the mutual enhancements between their bioconversions by catalyzing TCPP dephosphorylation and facilitating Pb 2+ bioprecipitation. TCPP may alleviate the Pb 2+ -induced oxidative stress by aiding protein phosphorylation. 80 % of Pb 2+ converted into crystalized pyromorphite. These results provide the mechanistic foundations and help develop greener strategies for synergistic bioremediation of OPEs and HMs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Aminoglycoside-modifying enzymes (AMEs) play an essential role in bacterial resistance to aminoglycoside antimicrobials. With the development of sequencing techniques, more bacterial genomes have been sequenced, which has aided in the discovery of an increasing number of novel resistance mechanisms., Methods: The bacterial species was identified by 16S rRNA gene homology and average nucleotide identity (ANI) analyses. The minimum inhibitory concentration (MIC) of each antimicrobial was determined by the agar dilution method. The protein was expressed with the pCold I vector in E. coli BL21, and enzyme kinetic parameters were examined. The whole-genome sequence of the bacterium was obtained via the Illumina and PacBio sequencing platforms. Reconstruction of the phylogenetic tree, identification of conserved functional residues, and gene context analysis were performed using the corresponding bioinformatic techniques., Results: A novel aminoglycoside resistance gene, designated aph(3')-Ie , which confers resistance to ribostamycin, kanamycin, sisomicin and paromomycin, was identified in the chromosome of the animal bacterium Citrobacter gillenii DW61, which exhibited a multidrug resistance phenotype. APH(3')-Ie showed the highest amino acid identity of 74.90% with the functionally characterized enzyme APH(3')-Ia. Enzyme kinetics analysis demonstrated that it had phosphorylation activity toward four aminoglycoside substrates, exhibiting the highest affinity ( K m , 4.22 ± 0.88 µM) and the highest catalytic efficiency [ k cat / K m , (32.27 ± 8.14) × 10 4 ] for ribomycin. Similar to the other APH(3') proteins, APH(3')-Ie contained all the conserved functional sites of the APH family. The aph(3')-Ie homologous genes were present in C. gillenii isolates from different sources, including some of clinical significance., Conclusion: In this work, a novel chromosomal aminoglycoside resistance gene, designated aph(3')-Ie , conferring resistance to aminoglycoside antimicrobials, was identified in a rabbit isolate C. gillenii DW61. The elucidation of the novel resistance mechanism will aid in the effective treatment of infections caused by pathogens carrying such resistance genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2024 Lin, Sha, Zhang, Song, Zhang, Zhao, Huang, Lu, Bao and Pan.)

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 β-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to β-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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tsypin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 (NH 4 ) 2 SO 4 with KNO 3 was the most preferred for achieving high growth density followed by (NH 4 ) 2 SO 4 and KNO 3 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 KNO 3 . 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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The study aims to see how effective the Citrobacter species strain is in removing Hg II under stressful conditions. For this, a response surface methodology was chosen to optimized pH, temperature, and biomass for effective biotransformation of Hg II . The optimized value for pH, temperature, and biomass were 6.5, 30 °C, and 2 mg/l with 89% Hg II 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 β-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 Hg II stress condition as well as promotion of Solanum lycopersicum L growth under stress conditions as a promising host., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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. K m and V max 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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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.

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., Competing Interests: YDL and TC are employees of Danisco Animal Nutrition. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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)-2β-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.

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 β-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.

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.

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.

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., Competing Interests: Competing interestsThe authors declare no competing interests.

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 C 33 N 4 O 9 H 36 S, which conformed to the predicted structure of bilirubin-10-sulfonate (BRS) and possessed a λ max 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.

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 N‑butyryl‑l‑homoserine lactone (C4-HSL), N‑hexanoyl‑l‑homoserine lactone (C6-HSL), N‑octanoyl‑l‑homoserine lactone (C8-HSL), N‑tetradecanoyl‑l‑homoserine lactone (C14-HSL) and N‑hexadecanoyl‑l‑homoserine 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2018. Published by Elsevier B.V.)

Roles of Fe 3 O 4 nanoparticles (NPs) on biodegradation of Paclitaxel by Citrobacter amalonaticus immobilized on alginate gel beads were investigated. Limitation in substrate diffusion is the major drawback of the cell immobilization method. To overcome this problem, bacterial cells were immobilized on the gel beads containing different concentrations (5-20mg/mL) of Fe 3 O 4 NPs and their Paclitaxel degrading potential at different temperatures was investigated using kinetic and thermodynamic modeling. Co-immobilization of bacterial cells with 5, 10 and 20mg/mL Fe 3 O 4 NPs enhanced biodegradation efficiencies to 66%, 80% and 78%, respectively, compared to the NPs free immobilized cells (41.9%). The optimum concentration of Fe 3 O 4 NPs (10mg/mL) had both inhibitory and accelerating effects on paclitaxel degradation depending on the incubation time and temperature. Increasing dose of Fe 3 O 4 NPs could increase paclitaxel degradation, despite increasing of thermodynamic inhibitory factors, only when longer time and higher temperature were used. ΔG values increased about 11.2 KJ/mol at all temperatures of 285, 295 and 305K, and ΔH increased 54.4%, in comparison with the treatment without NPs. This indicates that, inclusion of Fe 3 O 4 NPs into the immobilization gels can increase the local concentration of Paclitaxel (with OH 2 + groups) and bacterial accessibility to the substrate and thus enhance biodegradation efficiency., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Typhimurium is the only organism demonstrated to utilize fructose-asparagine (F-Asn) as a source of carbon and nitrogen. In this report, we first used a bioinformatics approach to identify other microorganisms that encode homologs of the Salmonella F-Asn utilization enzymes FraB (deglycase), FraD (kinase), and FraE (asparaginase). These candidate organisms were then tested with up to four different methods to confirm their ability to utilize F-Asn. The easiest and most broadly applicable method utilized a biological toxicity assay, which is based on the observation that F-Asn is toxic to a Salmonella fraB mutant. Candidate organisms were grown in a rich medium containing F-Asn, and depletion of F-Asn from the medium was inferred by the growth of a Salmonella fraB mutant in that same medium. For select organisms, the toxicity assay was cross-validated by direct mass spectrometry-aided measurement of F-Asn in the spent-culture media and through demonstration of FraB and FraD enzyme activity in cellular extracts. For prototrophs, F-Asn utilization was additionally confirmed by growth in a minimal medium containing F-Asn as the sole carbon source. Collectively, these studies established that Clostridium bolteae , Clostridium acetobutylicum , and Clostridium clostridioforme can utilize F-Asn, but Clostridium difficile cannot; Klebsiella oxytoca and some Klebsiella pneumoniae subspecies can utilize F-Asn; and some Citrobacter rodentium and Citrobacter freundii strains can also utilize F-Asn. Within Salmonella enterica , the host-adapted serovars Typhi and Paratyphi A have lost the ability to utilize F-Asn. IMPORTANCE Fructose-asparagine (F-Asn) is a precursor to acrylamide that is found in human foods, and it is also a nutrient source for Salmonella enterica , a foodborne pathogen. Here, we determined that among the normal intestinal microbiota, there are species of Clostridium that encode the enzymes required for F-Asn utilization. Using complementary experimental approaches, we have confirmed that three members of Clostridium , two members of Klebsiella , and two members of Citrobacter can indeed utilize F-Asn. The Clostridium spp. likely compete with Salmonella for F-Asn in the gut and contribute to competitive exclusion. FraB, one of the enzymes in the F-Asn utilization pathway, is a potential drug target because inhibition of this enzyme leads to the accumulation of a toxic metabolite that inhibits the growth of Salmonella species. This study identifies the potential off-target organisms that need to be considered when developing therapeutics directed at FraB., (Copyright © 2018 American Society for Microbiology.)

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.

Trinitrotoluene (TNT) is an explosive chemical generally used for military, civil and industrial purposes. Therefore, TNT residue can be found in soil and ground water as an environmental pollutant. The environmental control of TNT pollution has become a critical issue because of its potential toxicity and carcinogenicity. The aim of this study is to evaluate the cyto-toxicological effects of TNT after bioremediation. Citrobacter sp. YC4 is able to utilize TNT as a sole nitrogen source. Citrobacter sp. YC4 cells grown in medium with TNT as the sole nitrogen source (TNT-N) were able to rapidly degrade TNT, in contrast to cells grown in Luria Bertani medium as determined by resting cell suspension. The concentration of TNT decreased from 100 to 0 ppm within 10 h in the solution containing TNT mixed with TNT-N-grown YC4. The cytotoxicity of TNT and its degradation products generated by TNT-N-grown YC4 were assessed by WST-1-based cell cytotoxicity assays. Our results showed that the cytotoxic potential of solutions containing TNT decreased almost to the level of the control after a 1-h incubation with TNT-N-grown YC4 cells. The rapid conversion of TNT into possibly less toxic products by Citrobacter sp. YC4 proposes a bioremediation prospection., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Microbial fuel cells (MFCs) have attracted intensive interest for their power generation and pollutants removal characteristics. Electrochemical performances and community structures of two algae cathode photosynthetic MFCs were investigated and compared. Microbial consortia of these two MFCs were taken from wetland sediment (named SMFC) and an up-flow anaerobic wastewater treatment reactor (named UMFC). Maximum power density of the SMFC and UMFC achieved 202.9 ± 18.1 mW/m 2 and 158.2±15.1 mW/m 2 , respectively. The SMFC displayed higher columbic efficiency but lower chemical oxygen demand (COD) removal efficiency than that of UMFC. The results also revealed the addition of riboflavin (RF) and neutral red (NR) decreased the redox current of the SMFC but promoted that of UMFC. Community structure analysis showed the SMFC was dominated by photosynthetic genus Rhodopseudomonas (61.25%), while bacterial genera in the UMFC were more evenly distributed. The difference of electrochemical activities of the two MFCs was caused by the different roles of exoelectrogens such as Rhodopseudomonas spp. and Citrobacter spp. in the electron transfer process. Newly developed photosynthetic microbial fuel cells (PMFCs) provide a suitable process to generate power and remove pollutants. The consortia have a significant role in the performance and microbial community of the system., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Dissimilatory arsenate-respiring prokaryotes (DARPs) play key roles in the mobilization and release of arsenic from mineral phase into groundwater; however, little is known about how environmental factors influence these processes. This study aimed to explore the effects of sulfate on the dissolution and release of insoluble arsenic. We collected high-arsenic sediment samples from different depths in Jianghan Plain. Microcosm assays indicated that the microbial communities from the samples significantly catalyzed the dissolution, reduction and release of arsenic and iron from the sediments. Remarkably, when sulfate was added into the microcosms, the microorganisms-mediated release of arsenic and iron was significantly increased. To further explore the mechanism of this finding, we isolated a novel DARP, Citrobacter sp. JH001, from the samples. Arsenic release assays showed that JH001 can catalyze the dissolution, reduction and release of arsenic and iron from the sediments, and the presence of sulfate in the microcosms also caused a significant increase in the JH001-mediated dissolution and release of arsenic and iron. Quantitative PCR analysis for the functional gene abundances showed that sulfate significantly increased the arsenate-respiring reductase gene abundances in the microcosms. Thus, it can be concluded that sulfate significantly enhances the arsenate-respiring bacteria-mediated arsenic contamination in groundwater., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, the bacterial strain Citrobacter youngae strain E4 was isolated from 2,4,6-trinitrotoluene (TNT)-contaminated soil and used to assess the capacity of TNT transformation with/without exogenous nutrient amendments. C. youngae E4 poorly degraded TNT without an exogenous amino nitrogen source, whereas the addition of an amino nitrogen source considerably increased the efficacy of TNT transformation in a dose-dependent manner. The enhanced TNT transformation of C. youngae E4 was mediated by increased cell growth and up-regulation of TNT nitroreductases, including NemA, NfsA and NfsB. This result indicates that the increase in TNT transformation by C. youngae E4 via nitrogen nutrient stimulation is a cometabolism process. Consistently, TNT transformation was effectively enhanced when C. youngae E4 was subjected to a TNT-contaminated soil slurry in the presence of an exogenous amino nitrogen amendment. Thus, effective enhancement of TNT transformation via the coordinated inoculation of the nutrient-responsive C. youngae E4 and an exogenous nitrogen amendment might be applicable for the remediation of TNT-contaminated soil. Although the TNT transformation was significantly enhanced by C. youngae E4 in concert with biostimulation, the 96-h LC50 value of the TNT transformation product mixture on the aquatic invertebrate Tigriopus japonicas was higher than the LC50 value of TNT alone. Our results suggest that exogenous nutrient amendment can enhance microbial TNT transformation; however, additional detoxification processes may be needed due to the increased toxicity after reduced TNT transformation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to characterize β-glucosidase from Citrobacter koser GXW-1 isolated from soil and to improve the enzyme by molecular modification., Mehods: A bacterial strain with β-glucosidase activity was screened from the soil around Wuming sugar mill in Guangxi by esculin-ferric ammonium citrate selecting plate. The 16S rDNA of the strain was obtained and analyzed. By searching GenBank database, the genes encoding β-glucosidase from the same genus Citrobacter were found. These sequences were aligned. Then, a gene encoding β-glucosidase was amplified by PCR. The recombinant plasmid pQE-cbgl was constructed. The recombinant protein was purified with Ni-NTA. The enzyme properties of the recombinant protein CBGL were studied in detail. At last, the wild enzyme CBGL was reformed by error-prone PCR and site-directed random mutagenesis., Results: C. koser GXW-1 with β-glucosidase activity was isolated from the soil. A gene encoding β-glucosidase was cloned from the wild strain GXW-1. The properties of CBGL were identified. Its optimal pH and temperature were 6.0 and 45℃. Its Km and Vmax value were (11.280±1.073) mmol/L and (0.1704±0.0073) μmol/(mg·min), respectively. Its Ki values was (66.84±3.40) mmol/L. CBGL can hydrolyze α-pNPG, stevioside, daidzin and genistin. CBGL was modified by error-prone PCR and site directed random mutagenesis. A positive mutant W147F was obtained successfully. Its Vmax was 2.54 times that of the wild enzyme CBGL., Conclusion: CBGL not only can hydrolyze β-glycosidic bond, but also can hydrolyze the α-glycosidic bond in α-pNPG. Furthermore, CBGL can hydrolyze stevioside, daidzin and genistin. These characteristics indicate that the β-glucosidase CBGL has important applications in theoretical research and in industry.

Recent reports describe the ability of select bacterial strains to bind human norovirus, although the specificity of such interactions is unknown. The purpose of this work was to determine if a select group of bacterial species representative of human gut microbiota bind to human norovirus, and if so, to characterize the intensity and location of that binding. The bacteria screened included naturally occurring strains isolated from human stool (Klebsiella spp., Citrobacter spp., Bacillus spp., Enterococcus faecium and Hafnia alvei) and select reference strains (Staphylococcus aureus and Enterobacter cloacae). Binding in PBS was evaluated to three human norovirus strains (GII.4 New Orleans 2009 and Sydney 2012, GI.6) and two surrogate viruses (Tulane virus and Turnip Crinkle Virus (TCV)) using a suspension assay format linked to RT-qPCR for quantification. The impact of different overnight culture media prior to washing on binding efficiency in PBS was also evaluated, and binding was visualized using transmission electron microscopy. All bacteria tested bound the representative human norovirus strains with high efficiency (<1 log10 of input virus remained unbound or <10% unbound and >90% binding efficiency) (p>0.05); there was selective binding for Tulane virus and no binding observed for TCV. Binding efficiency was highest when bacteria were cultured in minimal media (<1 log10 of input virus remained unbound, so >90% bound), but notably decreased when cultured in enriched media (1-3 log10 unbound or 0.01 -<90% bound)) (p<0.05). The norovirus-bacteria binding occurred around the outer cell surfaces and pili structures, without apparent localization. The findings reported here further elucidate and inform the dynamics between human noroviruses and enteric bacteria with implications for norovirus pathogenesis.

Bioflocculation, being environmental-friendly and highly efficient, is considered to be a promising method to harvest microalgae. However, one limitation of this technology is high expense on substrates for bioflocculant bacteria cultivation. In this regard, we developed an innovative method for the inner-recycling of biomass that could harvest the typical microalgae, Microcystis aeruginosa, using a bioflocculant produced by Citrobacter sp. AzoR-1. In turn, the flocculated algal biomass could be reutilized as a substrate for Citrobacter sp. AzoR-1 cultivation and bioflocculant production. The experimental results showed that 3.4 ± 0.1 g of bioflocculant (hereafter called MBF-12) was produced by 10 g/L of wet biomass of M. aeruginosa (high-pressure steam sterilized) with an additional 10 g/L of glucose as an extra carbon source. The efficiency of MBF-12 for M. aeruginosa harvesting could reach ~95% under the optimized condition. Further analysis showed that MBF-12, dominated by ~270 kDa biopolymers, contributed the bioflocculation mechanisms of interparticle bridging and biosorption process. Bioflocculant synthesis by Citrobacter sp. AzoR-1 using microalga as a substrate, including the polyketide sugar unit, lipopolysaccharide, peptidoglycan and terpenoid backbone pathways. Our research provides the first evidence that harvested algae can be reutilized as a substrate to grow a bioflocculant using Citrobacter sp. AzoR-1.

Hydrogen was produced from steam-exploded corn stover by using a combination of the cellulolytic bacterium Clostridium cellulolyticum and non-cellulolytic hydrogen-producing bacteria. The highest hydrogen yield of the co-culture system with C. cellulolyticum and Citrobacter amalonaticus reached 51.9 L H2/kg total solid (TS). The metabolites from the co-culture system were significantly different from those of the mono-culture systems. Formate, which inhibits the growth of C. cellulolyticum, could be consumed by the hydrogen-evolving bacteria, and transformed into hydrogen. Glucose and xylose were released from corn stover via hydrolysis by C. cellulolyticum and were quickly utilized in dark fermentation with the co-cultured hydrogen-producing bacteria. Because the hydrolysis of corn stover by C. cellulolyticum was much slower than the utilization of glucose and xylose by the hydrogen-evolving bacteria, the sugar concentrations were always maintained at low levels, which favored a high hydrogen molar yield., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Heavy metal-resistant bacteria can be efficient bioremediators of metals and may provide an alternative or additional method to conventional methods of metal removal. In this study, 10 bacterial isolates were isolated from soil samples of a sugar industry, located at Peshawar, Pakistan. Morphological, physiological, and biochemical characteristics of these isolates were observed. Sequence analysis (16S ribosomal RNA) revealed that isolated strains were closely related to the species belonging to the genera Pseudomonas, Arthrobacter, Exiguobacterium, Citrobacter, and Enterobacter Bacterial isolates were resistant with a minimum inhibitory concentration (500-900 ppm) to lead ion (Pb(2+)), (500-600 ppm) nickel ion (Ni(2+)), (500-800 ppm) copper ion (Cu(2+)), and (600-800 ppm) chromium ion (Cr(3+)) in solid media. Furthermore, biosorption of metals proved considerable removal of heavy metals by isolated metal-resistant strains. Pseudomonas sp. reduced 37% (Pb(2+)), 32% (Ni(2+)), 29% (Cu(2+)), and 32% (Cr(3+)) and was thus found to be most effective, whereas Enterobacter sp. reduced 19% (Pb(2+)), 7% (Ni(2+)), 14% (Cu(2+)), and 21% (Cr(3+)) and was found to be least effective. While average reduction of Pb(2+), Ni(2+), Cu(2+), and Cr(3+) by Citrobacter sp. was found to be 24%, 18%, 23%, and 27%, respectively, among recognized species. This study revealed that Pseudomonas sp. may provide a new microbial community that can be used for enhanced remediation of contaminated environment., (© The Author(s) 2015.)

Paclitaxel is a highly toxic anticancer agent which is used in a wide range against ovarian, breast, lung, and prostate cancers. Paclitaxel is manufactured recently in the north of Iran which may lead to the introduction of the drug into the environment via pharmaceutical wastewater. To our knowledge, Paclitaxel degradation is currently performed using physicochemical methods and biological degradation of Paclitaxel has not been reported. In this study, a Paclitaxel degrading bacterium was isolated from pharmaceutical wastewater for the first time. The bacterium was identified using biochemical and molecular assays and its Paclitaxel degradation potential was evaluated using High Performance Liquid Chromatography (HPLC). In addition, kinetic and thermodynamic study of Paclitaxel degradation at different experimental conditions was performed. A Citrobacter species named as C. amalonaticus Rashtia able to degrade and utilize Paclitaxel as the sole carbon source was isolated. The isolated strain tolerated high level concentration of Paclitaxel (0.4 mg/mL) in liquid culture media and was able to degrade spillage-level concentrations of the drug (0.01-0.1 mg/mL) with 87-93 % efficacy under aerobic condition. Kinetic and thermodynamic study at different pHs (4.0, 7.0 and 10.0) and temperatures (285, 295 and 310 K) revealed that Paclitaxel degradation is a non-spontaneous process and the highest rate constant was observed in the basic condition and at the highest temperature. The ΔG values at 285, 295 and 310 K were determined 103.3, 105.9 and 109.9 kJ/mol, respectively. In addition, The ΔH and activation energy (Ea) of the process were determined +28.7 kJ/mol and +30.87 kJ/mol, respectively.

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads' superior resilience compared to E. coli. Whole-cell P. putida harboring P450cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield YP/S of 79 %. To the authors' knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.

Selective enrichment of cathodic bacterial community was investigated during reductive decolorization of AYR fedding with glucose or acetate as co-substrates in biocathode. A clear distinction of phylotype structures were observed between glucose-fed and acetate-fed biocathodes. In glucose-fed biocathode, Citrobacter (29.2%), Enterococcus (14.7%) and Alkaliflexus (9.2%) were predominant, and while, in acetate-fed biocathode, Acinetobacter (17.8%) and Achromobacter (6.4%) were dominant. Some electroactive or reductive decolorization genera, like Pseudomonas, Delftia and Dechloromonas were commonly enriched. Both of the higher AYR decolorization rate (k(AYR)=0.46) and p-phenylenediamine (PPD) generation rate (k(PPD)=0.38) were obtained fed with glucose than acetate (k(AYR)=0.18; k(PPD)=0.16). The electrochemical behavior analysis represented a total resistance in glucose-fed condition was about 73.2% lower than acetate-fed condition. The different co-substrate types, resulted in alteration of structure, richness and composition of bacterial communities, which significantly impacted the performances and electrochemical behaviors during reductive decolorization of azo dyes in biocathode., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Restriction-modification (R-M) systems are highly prevalent among bacteria and archaea, and appear to play crucial roles in modulating horizontal gene transfer and protection against phage. There is much to learn about these diverse enzymes systems, especially their regulation. Type II R-M systems specify two independent enzymes: a restriction endonuclease (REase) and protective DNA methyltransferase (MTase). Their activities need to be finely balanced in vivo Some R-M systems rely on specialized transcription factors called C (controller) proteins. These proteins play a vital role in the temporal regulation of R-M gene expression, and function to indirectly modulate the horizontal transfer of their genes across the species. We report novel regulation of a C-responsive R-M system that involves a C protein of a poorly-studied structural class - C.Csp231I. Here, the C and REase genes share a bicistronic transcript, and some of the transcriptional auto-control features seen in other C-regulated R-M systems are conserved. However, separate tandem promoters drive most transcription of the REase gene, a distinctive property not seen in other tested C-linked R-M systems. Further, C protein only partially controls REase expression, yet plays a role in system stability and propagation. Consequently, high REase activity was observed after deletion of the entire C gene, and cells bearing the ΔC R-M system were outcompeted in mixed culture assays by those with the WT R-M system. Overall, our data reveal unexpected regulatory variation among R-M systems., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The spent fluorescent lamps (SFLs) are being classified as a hazardous waste due to having mercury as one of its main components. Mercury is considered the second most toxic heavy metal (arsenic is the first) with harmful effects on animal nervous system as it causes different neurological disorders. In this research, the mercury from phosphor powder was leached, then bioremediated using bacterial strains isolated from Qatari environment. Leaching of mercury was carried out with nitric and hydrochloric acid solutions using two approaches: leaching at ambient conditions and microwave-assisted leaching. The results obtained from this research showed that microwave-assisted leaching method was significantly better in leaching mercury than the acid leaching where the mercury leaching efficiency reached 76.4%. For mercury bio-uptake, twenty bacterial strains (previously isolated and purified from petroleum oil contaminated soils) were sub-cultured on Luria Bertani (LB) plates with mercury chloride to check the bacterial tolerance to mercury. Seven of these twenty strains showed a degree of tolerance to mercury. The bio-uptake capacities of the promising strains were investigated using the mercury leached from the fluorescent lamps. Three of the strains (Enterobacter helveticus, Citrobacter amalonaticus, and Cronobacter muytjensii) showed bio-uptake efficiency ranged from 28.8% to 63.6%., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cell-cell communication is also known as quorum sensing (QS) that happens in the bacterial cells with the aim to regulate their genes expression in response to increased cell density. In this study, a bacterium (L8A) isolated from dental plaque biofilm was identified as Citrobacter amalonaticus by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Its N-acylhomoserine-lactone (AHL) production was screened by using two types of AHL biosensors namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Citrobacter amalonaticus strain L8A was identified and confirmed producing numerous types of AHL namely N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexadecanoyl-L-homoserine lactone (C16-HSL). We performed the whole genome sequence analysis of this oral isolate where its genome sequence reveals the presence of QS signal synthase gene and our work will pave the ways to study the function of the related QS genes in this bacterium.

Background: Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii ∆dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply., Results: By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA, and ethanol dehydrogenase coded by adhE) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii ∆dhaD resulted in the strain C. werkmanii ∆dhaD∆ldhA∆adhE::ChlFRT which provided the maximum theoretical yield of 1.00 ± 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 ± 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism., Conclusions: In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached.

We report here the complete genome sequence of Citrobacter amalonaticus Y19 isolated from an anaerobic digester. PacBio single-molecule real-time (SMRT) sequencing was employed, resulting in a single scaffold of 5.58Mb. The sequence of a mega plasmid of 291Kb size is also presented., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To gain insights into the diversification trajectories of qnrB genes, a phylogenetic and comparative genomics analysis of these genes and their surrounding genetic sequences was performed. For this purpose, Citrobacter sp. isolates (n = 21) and genome or plasmid sequences (n = 56) available in public databases harboring complete or truncated qnrB genes were analyzed. Citrobacter species identification was performed by phylogenetic analysis of different genotypic markers. The clonal relatedness among isolates, the location of qnrB genes, and the genetic surroundings of qnrB genes were investigated by pulsed-field gel electrophoresis (PFGE), S1-/I-CeuI-PFGE and hybridization, and PCR mapping and sequencing, respectively. Identification of Citrobacter isolates was achieved using leuS and recN gene sequences, and isolates characterized in this study were diverse and harbored chromosomal qnrB genes. Phylogenetic analysis of all known qnrB genes revealed seven main clusters and two branches, with most of them included in two clusters. Specific platforms (comprising pspF and sapA and varying in synteny and/or identity of other genes and intergenic regions) were associated with each one of these qnrB clusters, and the reliable identification of all Citrobacter isolates revealed that each platform evolved in different recognizable (Citrobacter freundii, C. braakii, C. werkmanii, and C. pasteurii) and putatively new species. A high identity was observed between some of the platforms identified in the chromosome of Citrobacter spp. and in different plasmids of Enterobacteriaceae. Our data corroborate Citrobacter as the origin of qnrB and further suggest divergent evolution of closely related qnrB genes/platforms in particular Citrobacter spp., which were delineated using particular genotypic markers., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In this study, we investigated the differences in histamine accumulation between blue scad and chub mackerel and methods of inhibiting histamine-forming bacteria and controlling histamine accumulation in fish. The free histidine contents in blue scad and chub mackerel were 1.45 and 2.75 mg/g, respectively. The histamine-forming bacteria isolated from them were identified as Citrobacter freundii, Citrobacter braakii, and Enterobacter aerogenes using 16S rDNA sequence analysis, the VITEK 2 Compact system, and MALDI-TOF MS. The histamine-producing capacities of C. freundii, C. braakii, and E. aerogenes were 470, 1,057, and 4,213 mg/liter, respectively, after culture at 37°C for 48 h. Among the different antimicrobials and preservatives tested, potassium sorbate and sodium diacetate effectively inhibited the histamine-forming bacteria and their histamine production. After chub mackerel was dipped into 0.5% potassium sorbate or sodium diacetate, its histamine content increased more slowly at room temperature. Therefore, a potassium sorbate or sodium diacetate dipping treatment could effectively control histamine accumulation in fish.

In a wide variety of bacterial restriction-modification systems, a regulatory `controller' protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.

Citrobacter sedlackii RI11, isolated from acclimated textile effluent after selective enrichment on synthetic dyes, was assessed for malachite green (MG) biotreatment potency. Results indicate that this bacterium has potential for use in effective treatment of MG contaminated wastewaters under shaking conditions at neutral and alkaline pH value, characteristic of typical textile effluents. Also, the newly isolated strain can tolerate higher doses of dye and decolorize up to 1,000 mg/l of dye. When used as microbial surfactant to enhance MG biodecolorization, Bacillus subtilis SPB1-derived lipopeptide accelerated the decolorization rate and maximized the decolorization efficiency at an optimal concentration of biosurfactant of about 0.075%. Studies ensured that MG removal by this strain could be due to biodegradation and/or adsorption. Results on germination potencies of different seeds using the treated dyes under different conditions favor the use of SPB1 biosurfactant for the treatment of MG.

To attain a better understanding of the effects of surfactants on the metabolic kinetics of hydrophobic organic compounds, the biodegradation of phenanthrene by Citrobacter sp. SA01 was investigated in a batch experiment containing Tween 80, sodium dodecyl benzene sulfonate and liquid mineral salt medium. The Monod model was modified to effectively describe the partition, phenanthrene biodegradation and biopolymer production. The results showed that Tween 80 and sodium dodecyl benzene sulfonate (each at 50mg/L) enhanced phenanthrene metabolism and poly-β-hydroxybutyrate production as indicated by the increasing amounts of intermediates (by 17.2% to 47.9%), and percentages of poly-β-hydroxybutyrate (by 107.3% and 33.1%) within the cell dry weight when compared to their absence. The modified Monod model was capable of predicting microbial growth, phenanthrene depletion and biopolymer production. Furthermore, the Monod kinetic coefficients were largely determined by the surfactant-enhanced partition, suggesting that partitioning is a critical process in surfactant-enhanced bioremediation of hydrophobic organic compounds., (Copyright © 2014. Published by Elsevier B.V.)

Biofilms contain a diverse range of microorganisms and their varying extracellular polysaccharides. The present study has revealed biofilm succession associated with degradative effects on plastic (polypropylene) and contaminants in sludge. The wet weight of biofilm significantly (p < 0.05) increased; from 0.23 ± 0.01 to 0.44 ± 0.01 g. Similarly, the dry weight of the biofilm increased from 0.02 to 0.05 g. Significant reduction in pathogens (E. coli and feacal coliforms) by MPN technique (>80%) and in chemical parameters (decrease in COD, BOD5 of 73.32 and 69.94%) representing diminution of organic pollutants. Energy dispersive X-ray spectroscopy (EDS) of plastic revealed carbon and oxygen contents, further surface analysis of plastic by scanning electron microscopy (SEM) revealed emergence of profound bacterial growth on the surface. Fourier transform infrared (FTIR) spectroscopy conforms its biotransformation under aerobic conditions after 8 weeks. New peaks developed at the region 1050 and 969 cm(-1) indicating CO and CC bond formation. Thus plastic with 6 weeks old aerobic biofilm (free of pathogens, max. weight, and OD, efficient COD & BOD removal ability) is suggested to be maintained in fixed biofilm reactors for wastewater treatment., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)