Evidence for existence of quorum sensing in a bioaugmented system by acylated homoserine lactone-dependent quorum quenching.

The introduction of a gene, strain, or microbial consortium into an indigenous bacterial population is known as bioaugmentation. This technique has been proposed as an effective strategy for accelerating and enhancing the removal of recalcitrant and toxic compounds during wastewater treatment. In this study, three types of reactors were used to test whether quorum sensing plays an important role in bioaugmented systems. Reverse transcriptase polymerase chain reaction showed that the inoculated strain, HF-1, successfully colonized in the bioaugmented reactor. Meanwhile, no HF-1 colonization was observed in the quorum-quenching and non-bioaugmented reactors. Removal of nicotine in the bioaugmented reactor was almost 100%, and removal of total organic carbon (TOC) was higher than 50%. However, less than 20% of nicotine and 30% of TOC was removed in quorum-quenching and non-bioaugmented reactors. Moreover, the release of acylated homoserine lactones reached the threshold for HF-1 biofilm formation in bioaugmented reactors but not in quorum-quenching or non-bioaugmented reactors. The addition of porcine kidney acylase I, a quenching reagent, to the quorum-quenching reactor hampered the colonization of HF-1. Together, these results demonstrate that quorum sensing plays an important role in HF-1 colonization of bioaugmented systems.

A new method for rapid construction of a Pseudomonas sp. HF-1 bioaugmented system: accelerating acylated homoserine lactones secretion by pH regulation.

Pseudomonas sp. HF-1 bioaugmented systems were operated to treat tobacco wastewater under pH 5.5 for three cycles and pH 8.0 for the rest, which was suitable for HF-1 biofilm formation. The results showed that, under pH control, the contents of 3-oxo-C6-HSL, C6-HSL and 3-oxo-C8-HSL were significantly higher than HF-1 thresholds for biofilm formation. Compared with non-pH controlled reactors, HF-1 showed greater colonization in pH controlled reactors, primarily owing to the high extracellular polymeric substances secretion induced by quorum sensing. Accordingly, high indigenous community activity and granular sludge were observed. Sludge granulation occurred from the seventh cycle, and the average diameter was greater than 400 µm. These systems were also highly efficient with nearly 100% nicotine degradation and 60% total organic carbon removal. Overall, the results indicate that pH regulation is a new and feasible method for acceleration of releasing of auto-inducers, which is beneficial to construction of HF-1 bioaugmented systems.

Effect of Pseudomonas sp. HF-1 inoculum on construction of a bioaugmented system for tobacco wastewater treatment: analysis from quorum sensing.

To better construct a bioaugmented system for tobacco wastewater treatment, activated sludge was inoculated with different concentrations of the nicotine-degrading bacterium Pseudomonas sp. HF-1. The results showed that inoculum concentrations of 0.55 ± 0.01 and 1.10 ± 0.03 mg/g (dry weight of strain HF-1/dry weight of activated sludge) were best to ensure strain HF-1 survival and successful bioaugmentation. The release pattern of autoinducer (AI) for quorum sensing in the bioaugmented system was also investigated. During the period of HF-1 inoculation, compared with failed bioaugmented systems, AI-2 was significantly increased in the successful systems, suggesting that AI-2-mediated bacterial communication played an important role in the colonization of HF-1. When inoculation of strain HF-1 was stopped, the amount of AI-2 decreased and leveled out in all systems. Notably, there was a greater than threefold increase of short-chain AHLs in failed bioaugmented systems, but no increase in successful ones, implying that the fluctuation of short-chain AHLs could be an indicator of the failure of bioaugmentation. Thus, AI-2-mediated quorum sensing could be implemented to facilitate HF-1 colonization.

Bioaugmentation of activated sludge with Acinetobacter sp. TW enhances nicotine degradation in a synthetic tobacco wastewater treatment system.

Bioaugmentation (BA) using Acinetobacter sp. TW with high nicotine-degrading efficiency was applied in a bioreactor receiving a load of COD (3,200 ± 50 mg/L) and nicotine (1.0 ± 0.1g/L). The results showed that because of the colonization of strain TW, the COD removal was stable at 80-90%, while nicotine removal reached 98% in the BA system. Furthermore, according to PCR-DGGE fingerprinting, compared with the originally activated sludge, more bacteria existed in the BA systems while some bacteria disappeared from the non-BA system. In terms of the quorum sensing, short chain AHLs increased to assist colonization of strain TW, and long chain AHLs were secreted and helped to resist the nicotine toxicity. Compared with the non-BA system, the amounts of ROS, protein carbonyls and 8-OHdG were significant lower in the BA systems, which suggested that strain TW played an important role in eliminating the nicotine toxicity from the bioreactors.

Ecological roles and release patterns of acylated homoserine lactones in Pseudomonas sp. HF-1 and their implications in bacterial bioaugmentation.

To enable development of a better bacterial bioaugmentation system for tobacco wastewater treatment, the roles and release patterns of acylated homoserine lactones (AHLs) in Pseudomonas sp. HF-1 were evaluated. Swarming was found to be induced by N-hexanoyl-homoserine lactone (C(6)-HSL) and N-3-oxo-hexanoyl-homoserine lactone (3-oxo-C(6)-HSL); the formation of extracellular polymeric substances (EPS) was induced by 3-oxo-C(6)-HSL, C(6)-HSL and N-3-oxo-octanoyl-homoserine lactone (3-oxo-C(8)-HSL); and biofilm formation was induced by C(6)-HSL and 3-oxo-C(8)-HSL. When the culture conditions were 25°C, pH 5-6, 3% inoculum, 1.5 g L(-1) nicotine and 1% NaCl, the amount of AHLs released was sufficient for quorum sensing of swarming and EPS formation for strain HF-1, which was beneficial to the startup stage during bioaugmentation. When strain HF-1 was cultured at pH 8 in the presence of 1.2-1.8 g L(-1) of nicotine and 1% NaCl, the threshold for quorum sensing of biofilm formation was reached and the bioaugmentation system showed an efficient performance.

Discovery of inhibitors of Escherichia coli methionine aminopeptidase with the Fe(II)-form selectivity and antibacterial activity.

Methionine aminopeptidase (MetAP) is a promising target to develop novel antibiotics, because all bacteria express MetAP from a single gene that carries out the essential function of removing N-terminal methionine from nascent proteins. Divalent metal ions play a critical role in the catalysis, and there is an urgent need to define the actual metal used by MetAP in bacterial cells. By high throughput screening, we identified a novel class of catechol-containing MetAP inhibitors that display selectivity for the Fe(II)-form of MetAP. X-ray structure revealed that the inhibitor binds to MetAP at the active site with the catechol coordinating to the metal ions. Importantly, some of the inhibitors showed antibacterial activity at low micromolar concentration on Gram-positive and Gram-negative bacteria. Our data indicate that Fe(II) is the likely metal used by MetAP in the cellular environment, and MetAP inhibitors need to inhibit this metalloform of MetAP effectively to be therapeutically useful.

(R)-albuterol decreases immune responses: role of activated T cells.

Racemic albuterol is an equimolar mixture of two isomers, (R) and (S). Whether (R) and (S) isomers and the combination of both exert different effects in immune activation is not well defined. We analyzed the effects of (R+S)-albuterol, (R)-albuterol and (S)-albuterol in a murine model of allergic pulmonary inflammation and in activated T cells. Mice (C57BL/6) sensitized and aerosol challenged with the allergen ovalbumin (OVA) or phosphate buffered saline (PBS) were treated with (R)-albuterol, (S)-albuterol or (R+S)-albuterol. Following administration of (R)-albuterol, allergen induced bronchoalveolar lavage eosinophils and IgE showed a decrease, albeit not significantly by ANOVA. As T cells are important in allergic inflammation, we asked whether (R+S), (R) or (S)-albuterol might differ in effects on T cells and on the activity of the inflammatory transcription factor NF-kappaB. In activated T cells, (R)-albuterol administration decreased levels of inflammatory cytokines and NF-kappaB activity. These studies suggest that (R)-albuterol decreases cytokine secretion and NF-kappaB activity in T cells.