Paracoccus benzoatiresistens sp. nov., a benzoate resistance and selenite reduction bacterium isolated from wetland.

A Gram-stain-negative, aerobic, non-motile, catalase- and oxidase-positive, pale orange, rod-shaped strain EF6T, was isolated from a natural wetland reserve in Hebei province, China. The strain grew at 25-37 °C (optimum, 30 °C), pH 5-9 (optimum, pH 7), and in the presence of 1.0-4.0% (w/v) NaCl (optimum, 2%). A phylogenetic analysis based on 16S rRNA gene sequence revealed that strain EF6T belongs to the genus Paracoccus, and the closest members were Paracoccus shandongensis wg2T with 98.1% similarity, Paracoccus fontiphilus MVW-1 T (97.9%), Paracoccus everestensis S8-55 T (97.7%), Paracoccus subflavus GY0581T (97.6%), Paracoccus sediminis CMB17T (97.3%), Paracoccus caeni MJ17T (97.0%), and Paracoccus angustae E6T (97.0%). The genome size of strain EF6T was 4.88 Mb, and the DNA G + C content was 65.3%. The digital DNA-DNA hybridization, average nucleotide identity, and average amino acid identity values between strain EF6T and the reference strains were all below the threshold limit for species delineation (< 32.8%, < 88.0%, and < 86.7%, respectively). The major fatty acids (≥ 5.0%) were summed feature 8 (86.3%, C18:1 ω6c and/or C18:1 ω7c) and C18:1 (5.0%) and the only isoprenoid quinone was Q-10. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, two unidentified glycolipids, five unidentified phospholipids, and an unidentified aminolipid. Strain EF6T displays notable resistance to benzoate and selenite, with higher tolerance levels (25 g/L for benzoate and 150 mM for selenite) compared to the closely related species. Genomic analysis identified six benzoate resistance genes (acdA, pcaF, fadA, pcaC, purB, and catA) and twenty selenite resistance and reduction-related genes (iscR, ssuB, ssuD, selA, selD and so on). Additionally, EF6T possesses unique genes (catA, ssuB, and ssuC) absent in the closely related species for benzoate and selenite resistance. Its robust resistance to benzoate and selenite, coupled with its genomic makeup, make EF6T a promising candidate for the remediation of both organic and inorganic pollutants. It is worth noting that the specific resistance phenotypes described above were not reported in other novel species in Paracoccus. Based on the results of biochemical, physiological, phylogenetic, and chemotaxonomic analyses, combined with comparisons of the 16S rRNA gene sequence and the whole genome sequence, strain EF6T is considered to represent a novel species of the genus Paracoccus within the family Rhodobacteraceae, for which the name Paracoccus benzoatiresistens sp. nov. is proposed. The type strain is EF6T (= GDMCC 1.3400 T = JCM 35642 T = MCCC 1K08702T).

A mediator microbial biosensor for assaying general toxicity.

A mediator biosensor based on Paracoccus yeei bacteria for assaying the toxicity of perfumery and cosmetics samples was developed. An approach to selecting an electron-transport mediator based on the heterogeneous electron transfer constants for investigated mediators (ks) and the mediator-biomaterial interaction constants (kinteract) was proposed. Screening of nine compounds as potential mediators showed a ferrocene mediator immobilized in graphite paste to have the highest efficiency of electron transfer to the graphite-paste electrode (the heterogeneous transfer constant, 0.4 ±â€¯0.1 cm/s) and a high constant of interaction with P. yeei (0.023 ±â€¯0.001 dm3/(g·s)). A biosensor for toxicity assessment based on the ferrocene mediator and P. yeei bacteria was formed. The biosensor was tested on samples of four heavy metals (Cu2+, Zn2+, Pb2+, Cd2+) and two phenols (phenol and p-nitrophenol). Proceeding from the EC50 index, it was found that the use of the ferrocene mediator made the biosensor more sensitive to investigated toxicants than most analogues described. Toxicity determination of four perfumery and cosmetics samples by the developed biosensor showed prospects of using this system for real-time toxicity monitoring of samples.

Characterization of the virome of Paracoccus spp. (Alphaproteobacteria) by combined in silico and in vivo approaches.

Bacteria of the genus Paracoccus inhabit various pristine and anthropologically-shaped environments. Many Paracoccus spp. have biotechnological value and several are opportunistic human pathogens. Despite extensive knowledge of their metabolic potential and genome architecture, little is known about viruses of Paracoccus spp. So far, only three active phages infecting these bacteria have been identified. In this study, 16 Paracoccus strains were screened for the presence of active temperate phages, which resulted in the identification of five novel viruses. Mitomycin C-induced prophages were isolated, visualized and their genomes sequenced and thoroughly analyzed, including functional validation of their toxin-antitoxin systems. This led to the identification of the first active Myoviridae phage in Paracoccus spp. and four novel Siphoviridae phages. In addition, another 53 prophages were distinguished in silico within genomic sequences of Paracoccus spp. available in public databases. Thus, the Paracoccus virome was defined as being composed of 66 (pro)phages. Comparative analyses revealed the diversity and mosaicism of the (pro)phage genomes. Moreover, similarity networking analysis highlighted the uniqueness of Paracoccus (pro)phages among known bacterial viruses.

Towards eco-friendly biocides: preparation, antibiofilm activity of hemibastadin analogues.

The antibiofilm activity of three hemibastadins analogues was evaluated against different marine bacterial strains through mono-species biofilms and through a multi-species model of biofilm. Results showed that compound 3 exhibited interesting antibiofilm efficiencies effective concentrations (EC50 ) in the range of 30-100 µmol l-1 without acute toxicity against bacteria. Toxicity against nontargeted organisms was also considered showing that the compound did not affect the global bacterial community at a concentration of 75-100 µmol l-1 . These results provided baseline data concerning the toxicity of antibiofilm biocides against marine organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports relevant information about antibiofilm activity of original derivatives of hemibastadin alkaloids. The most active compound was shown to act as a specific anti-biofilm inhibitor without affecting viability of the targeted bacteria no more than those of the global bacterial community of a seawater sample. Taken together, these findings indicate the potentiality of such compounds to be used as original nonbiocidal molecules for designing eco-friendly antifouling solutions.

Disruption of N-acyl-homoserine lactone-specific signalling and virulence in clinical pathogens by marine sponge bacteria.

In recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries. A combination of unique physicochemical properties and spatial niche-specific substrates, in wide-ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel bioactivities. One such area of ongoing research is the discovery of compounds that interfere with the cell-cell signalling process called quorum sensing (QS). Described as the next generation of antimicrobials, these compounds can target virulence and persistence of clinically relevant pathogens, independent of any growth-limiting effects. Marine sponges are a rich source of microbial diversity, with dynamic populations in a symbiotic relationship. In this study, we have harnessed the QS inhibition (QSI) potential of marine sponge microbiota and through culture-based discovery have uncovered small molecule signal mimics that neutralize virulence phenotypes in clinical pathogens. This study describes for the first time a marine sponge Psychrobacter sp. isolate B98C22 that blocks QS signalling, while also reporting dual QS/QSI activity in the Pseudoalteromonas sp. J10 and ParacoccusJM45. Isolation of novel QSI activities has significant potential for future therapeutic development, of particular relevance in the light of the pending perfect storm of antibiotic resistance meeting antibiotic drug discovery decline.

Influencing mechanisms of hematite on benzo(a)pyrene degradation by the PAH-degrading bacterium Paracoccus sp. Strain HPD-2: insight from benzo(a)pyrene bioaccessibility and bacteria activity.

Iron oxides are reactive inorganic soil components that play an important role in the fate and transport of organic pollutants. Here, hematite was selected to investigate its effect on the biodegradation of benzo[a]pyrene (BaP) by Paracoccus sp. strain HPD-2. Approximately 60% of the total BaP was degraded in the absence of hematite after 7 days but only 30.8 and 20.8% of that was degraded after the addition of 10 and 20 mg  mL-1 hematite, respectively, indicating that the addition of hematite could significantly inhibit the biodegradation of BaP (P < 0.05). The hematite also lowered bacterium activity by coating the cells and by generating reactive oxygen species that destroyed the cells. Two-photon confocal laser scanning microscope images showed that the addition of hematite substantially decreased the amount of BaP combined with the bacterium, and this also enabled us to observe directly the migration and regression of BaP in the interaction between HPD-2 and hematite. Higher death ratio of HPD-2 might lower the BaP access to live cells because dead cells have a higher adsorption affinity for BaP than live cells. These observations enhance our understanding of the mechanisms by which metal oxides, organic pollutants and degrading-bacteria interact during the biodegradation process.

Towards smart biocide-free anti-biofilm strategies: Click-based synthesis of cinnamide analogues as anti-biofilm compounds against marine bacteria.

A set of triazole-based analogues of N-coumaroyltyramine was designed to discover potential leads that may help in the control of bacterial biofilms. the most potent compounds act as inhibitors of biofilm development with EC50 closed to ampicillin (EC50 = 11 µM) without toxic effect on bacterial growth even at high concentrations(100 µM).

Evaluation of Sulfadiazine Degradation in Three Newly Isolated Pure Bacterial Cultures.

This study is aimed to assess the biodegradation of sulfadiazine (SDZ) and characterization of heavy metal resistance in three pure bacterial cultures and also their chemotactic response towards 2-aminopyrimidine. The bacterial cultures were isolated from pig manure, activated sludge and sediment samples, by enrichment technique on SDZ (6 mg L-1). Based on the 16S rRNA gene sequence analysis, the microorganisms were identified within the genera of Paracoccus, Methylobacterium and Kribbella, which were further designated as SDZ-PM2-BSH30, SDZ-W2-SJ40 and SDZ-3S-SCL47. The three identified pure bacterial strains degraded up to 50.0, 55.2 and 60.0% of SDZ (5 mg L-1), respectively within 290 h. On the basis of quadrupole time-of-flight mass spectrometry and high performance liquid chromatography, 2-aminopyrimidine and 4-hydroxy-2-aminopyrimidine were identified as the main intermediates of SDZ biodegradation. These bacteria were also able to degrade the metabolite, 2-aminopyrimidine, of the SDZ. Furthermore, SDZ-PM2-BSH30, SDZ-W2-SJ40 and SDZ-3S-SCL47 also showed resistance to various heavy metals like copper, cadmium, chromium, cobalt, lead, nickel and zinc. Additionally, all three bacteria exhibited positive chemotaxis towards 2-aminopyrimidine based on the drop plate method and capillary assay. The results of this study advanced our understanding about the microbial degradation of SDZ, which would be useful towards the future SDZ removal in the environment.

Screening of bromotyramine analogues as antifouling compounds against marine bacteria.

Rapid and efficient synthesis of 23 analogues inspired by bromotyramine derivatives, marine natural products, by means of CuSO4-catalysed [3+2] alkyne-azide cycloaddition is described. The final target was then assayed for anti-biofilm activity against three Gram-negative marine bacteria, Pseudoalteromonas ulvae (TC14), Pseudoalteromonas lipolytica (TC8) and Paracoccus sp. (4M6). Most of the synthesised bromotyramine/triazole derivatives are more active than the parent natural products Moloka'iamine (A) and 3,5-dibromo-4-methoxy-ß-phenethylamine (B) against biofilm formation by the three bacterial strains. Some of these compounds were shown to act as non-toxic inhibitors of biofilm development with EC50 < 200 µM without any effect on bacterial growth even at high concentrations (200 µM).

Nanomechanical properties of the sea-water bacterium Paracoccus seriniphilus--a scanning force microscopy approach.

The measurement of force-distance curves on a single bacterium provides a unique opportunity to detect properties such as the turgor pressure under various environmental conditions. Marine bacteria are very interesting candidates for the production of pharmaceuticals, but are only little studied so far. Therefore, the elastic behavior of Paracoccus seriniphilus, an enzyme producing marine organism, is presented in this study. After a careful evaluation of the optimal measurement conditions, the spring constant and the turgor pressure are determined as a function of ionic strength and pH. Whereas the ionic strength changes the turgor pressure passively, the results give a hint that the change to acidic pH increases the turgor pressure by an active mechanism. Furthermore, it could be shown, that P. seriniphilus has adhesive protrusions outside its cell wall.

Cleaning of titanium substrates after application in a bioreactor.

Plain and microstructured cp-titanium samples were studied as possible biofilm reactor substrates. The biofilms were grown by exposition of the titanium samples to bacteria in a flow cell. As bacteria the rod shaped gram negative Pseudomonas fluorescens and the spherical gram negative Paracoccus seriniphilus were chosen. Afterward, the samples were cleaned in subsequent steps: First, with a standard solvent based cleaning procedure with acetone, isopropanol, and ultrapure water and second by oxygen plasma sputtering. It will be demonstrated by means of x-ray photoelectron spectroscopy, fluorescence microscopy, and confocal laser scanning microscopy that oxygen plasma cleaning is a necessary and reliant tool to fully clean and restore titanium surfaces contaminated with a biofilm. The microstructured surfaces act beneficial to biofilm growth, while still being fully restorable after biofilm contamination. Scanning electron microscopy images additionally show, that the plasma process does not affect the microstructures. The presented data show the importance of the cleaning procedure. Just using solvents does not remove the biofilm and all its components reliably while a cleaning process by oxygen plasma regenerates the surfaces.

Chronic impact of sulfamethoxazole on acetate utilization kinetics and population dynamics of fast growing microbial culture.

The study evaluated the chronic impact of sulfamethoxazole on metabolic activities of fast growing microbial culture. It focused on changes induced on utilization kinetics of acetate and composition of the microbial community. The experiments involved a fill and draw reactor, fed with acetate and continuous sulfamethoxazole dosing of 50 mg/L. The evaluation relied on model evaluation of the oxygen uptake rate profiles, with parallel assessment of microbial community structure by 454-pyrosequencing. Continuous sulfamethoxazole dosing inflicted a retardation effect on acetate utilization in a way commonly interpreted as competitive inhibition, blocked substrate storage and accelerated endogenous respiration. A fraction of acetate was utilized at a much lower rate with partial biodegradation of sulfamethoxazole. Results of pyrosequencing with a replacement mechanism within a richer more diversified microbial culture, through inactivation of vulnerable fractions in favor of species resistant to antibiotic, which made them capable of surviving and competing even with a slower metabolic response.

Influence of DMF-induced oxidative stress on membrane and periplasmic proteins in Paracoccus sp. SKG.

The present study describes the N,N-dimethylformamide (DMF)-induced oxidative stress in Paracoccus sp. SKG. The oxidative stress was evaluated by analysing membrane and periplasmic proteins and K+ efflux, as well as by monitoring the activities of antioxidant enzymes like catalase, superoxide dismutase (SOD) and glutathione S-transferase (GST). The exposure of bacterial cells to a higher concentration of DMF resulted in the modification of membrane fatty acid composition which is accompanied by K+ efflux. Further, this oxidative stress resulted in increased periplasmic protein which can be attributed to the induction of GST and methionine sulphoxide reductase (Msr) enzymes under solvent stress. Paracoccus sp. SKG is tolerant to high concentrations of DMF up to 6% (v/v) and its toxic effects. DMF concentration-dependent induction of GST and Msr activities advocates the significant role of these enzymes in the bacterial defence system. The present study provides information which helps us to understand the ROS scavenging machinery in bacteria. The high tolerance of Paracoccus sp. SKG to DMF can be efficiently explored for various bioremediation and biotransformation applications.

Influence of zero-valent iron nanoparticles on nitrate removal by Paracoccus sp.

Nitrate contamination in drinking water is a major threat to public health. This study investigated the efficiency of denitrification of aqueous solutions in the co-presence of synthesized nanoscale zero-valent iron (nZVI; diameter: 20-80 nm) and a previously isolated Paracoccus sp. strain YF1. Various influencing factors were studied, such as oxygen, pH, temperature, and anaerobic corrosion products (Fe(2+), Fe(3+) and Fe3O4). With slight toxicity to the strain, nZVI promoted denitrification efficiency by providing additional electron sources under aerobic conditions. For example, 50 mg L(-1) nZVI increased the nitrate removal efficiency from 66.9% to 85.2%. However, a high concentration of nZVI could lead to increased production of Fe(2+), a toxic ion which could compromise the removal efficiency. Kinetic studies suggest that denitrification by both free cells, and nZVI-amended cells fitted well to the zero-order model. Temperature and pH are the major factors affecting nitrate removal and cell growth, with or without the presence of nZVI. In this study, nitrate removal and cell growth increased in the pH range of 6.5-8.0, and temperature range of 25-35 °C. These conditions favor the growth of the strain, which dominated denitrification in all scenarios involved. As for anaerobic corrosion products, compared with Fe(2+) and Fe(3+), Fe3O4 promoted denitrification by serving as an electron donor. Finally, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) confirmed attachments of nZVI on the surface of the cell, and the formation of iron oxides. This study indicated that, as an electron donor source with minimal cellular toxicity, nZVI could be used to promote denitrification efficiency under biotic conditions.

Determining the ecological impacts of organic contaminants in biosolids using a high-throughput colorimetric denitrification assay: a case study with antimicrobial agents.

Land application accounts for ∼ 50% of wastewater solid disposal in the United States. Still, little is known regarding the ecological impacts of nonregulated contaminants found in biosolids. Because of the myriad of contaminants, there is a need for a rapid, high-throughput method to evaluate their ecotoxicity. Herein, we developed a novel assay that measures denitrification inhibition in a model denitrifier, Paracoccus denitrificans Pd1222. Two common (triclosan and triclocarban) and four emerging (2,4,5 trichlorophenol, 2-benzyl-4-chlorophenol, 2-chloro-4-phenylphenol, and bis(5-chloro-2-hydroxyphenyl)methane) antimicrobial agents found in biosolids were analyzed. Overall, the assay was reproducible and measured impacts on denitrification over 3 orders of magnitude exposure. The lowest observable adverse effect concentrations (LOAECs) were 1.04 µM for triclosan, 3.17 µM for triclocarban, 0.372 µM for bis-(5-chloro-2-hydroxyphenyl)methane, 4.89 µM for 2-chloro-4-phenyl phenol, 45.7 µM for 2-benzyl-4-chorophenol, and 50.6 µM for 2,4,5-trichlorophenol. Compared with gene expression and cell viability based methods, the denitrification assay was more sensitive and resulted in lower LOAECs. The increased sensitivity, low cost, and high-throughput adaptability make this method an attractive alternative for meeting the initial testing regulatory framework for the Federal Insecticide, Fungicide, and Rodenticide Act, and recommended for the Toxic Substances Control Act, in determining the ecotoxicity of biosolids-derived emerging contaminants.

The response of Paracoccus sp. SKG to acetonitrile-induced oxidative stress.

Organic solvents enhance intracellular oxidative stress and induce various physiological responses in bacteria. The study shows the morphological changes in Paracoccus sp. SKG when exposed to higher concentrations of acetonitrile, which alter the composition of the membrane fatty acid that accompanies the increase in K(+) efflux. This enhances the oxidative stress with greater activities of catalase and super oxide dismutase (SOD). The increased oxidative stress results in the generation of free radicals, which was confirmed by electron paramagnetic resonance (EPR) studies. The free radical scavenging activities were measured by ABTS and DPPH to understand the non-enzymatic defensive system during oxidative stress. The studies demonstrate the increase in free radicals in association with enzymatic and non-enzymatic defense systems under solvent stress.

Impact of iron-based nanoparticles on microbial denitrification by Paracoccus sp. strain YF1.

This study investigates the effects of Fe and Fe/Ni nanoparticles on biological denitrification when using Paracoccus sp. strain YF1. Results show that adding Fe and Fe/Ni nanoparticles to the cells decreased their growth and denitrification rate. Compared to that of free cells (control 89.47%), a decrease (64.33%) in the presence of 1000 mg/L Fe/Ni nanoparticles was observed, while a small decline in the denitrification rate (76.36%) was obtained when the concentration of Fe nanoparticles was 1000 mg/L. These were further confirmed by adding Fe(2+), Fe(3+), Fe3O4, Fe(2+)/Ni(2+) and Fe(3+)/Ni(2+) individually to the free cell system. Furthermore, Fe and Fe/Ni nanoparticles influenced the nitrate removal and bacterial growth under different pH and temperature conditions. SEM, XRD and EDS demonstrated that iron oxides formed as a result of nanoparticles corrosion in biological medium. Finally the presence of nanoparticles around some bacteria was observed.

Biological removal of nitrate and ammonium under aerobic atmosphere by Paracoccus versutus LYM.

The bacterium isolated from sea sludge Paracoccus versutus LYM was characterized with the ability of aerobic denitrification. Strain LYM performs perfect activity in aerobically converting over 95% NO3(-)-N (approximate 400mg L(-1)) to gaseous products via nitrite with maximum reduction rate 33 mg NO3(-)-N L(-1) h(-1). Besides characteristic of aerobic denitrification, strain LYM was confirmed in terms of the ability to be heterotrophic nitrification and aerobic denitrification (HNAD) with few accumulations of intermediates. After the nitrogen balance and enzyme assays, the putative nitrogen pathway of HNAD could be NH4(+) → NH2OH → NO2(-)→ NO3(-), then NO3(-) was denitrified to gaseous products via nitrite. N2 was sole denitrification product without any detection of N2O by gas chromatography. Strain LYM could also simultaneously remove ammonium and additional nitrate. Meanwhile, the accumulated nitrite had inhibitory effect on ammonium reduction rate.