Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterial pathogen, well known for its remarkable ability to live and survive in diverse environments. The pathogen produces a range of virulence factors, such as exotoxin A, exoenzyme S, pyocyanin, proteases, elastase, rhamnolipids, and lipopolysaccharides, and frequently causes acute and chronic infections in immunocompromised hosts. In addition, P. aeruginosa may switch from a planktonic growth mode to a surface-attached lifestyle, i.e., biofilms, in response to biotic or abiotic stresses (14). Biofilm bacterial cells are stuck to each other and embedded in a self-manufactured matrix of extracellular polymeric substance, enabling them to escape from human defense responses and withstand high-dose antibiotic treatments. P. aeruginosa has become a serious concern in intensive care units, largely due to its biofilm-related drug resistance and the potential of biofilm as a source of contamination (16,41,43,46). Biofilm formation by P. aeruginosa progresses through multiple developmental stages, beginning with attachment to a surface, followed by migration and division to form microcolonies, and then maturation involving expression of matrix polymers. The biofilm developmental life cycle comes full circle when the biofilm cells disperse (51). For the convenience of discussion, we define here that biofilm development covers two phases, i.e., formation and dispersal. Recent research has revealed a range of factors associated with biofilm dispersal, including matrix-degrading enzymes (5), activation ...

Pseudomonas aeruginosa can grow either as planktonic- or biofilm-form in response to environmental changes. Recent studies show that switching from biofilm to planktonic lifestyle requires rhamnolipids. Here we report the identification of a novel twocomponent system BqsS-BqsR that regulates biofilm decay in P. aeruginosa. BqsS is a multidomain sensor kinase and BqsR is an OmpR-like response regulator. Deletion of either bqsS or bqsR in P. aeruginosa mPAO1 resulted in a significant increase in biofilm formation. Time course analysis showed that the bqsS-bqsR mutants were defective in biofilm dispersal and in rhamnolipid production. Mutation of the BqsS-BqsR two-component system did not affect the biosynthesis of long chain quorum sensing (QS) signal N-3-oxo-dodecanoyl-homoserine lactone (3OC12HSL) but resulted in reduced production of the short chain QS signal N-butyryl-L-homoserine lactone (C4HSL) and the Pseudomonas quinolone signal (PQS). Exogenous addition of C4HSL, PQS or rhamnolipids to the bqsS mutant reduced the biofilm formation to the wild-type level. Evidence suggests that the BqsS-BqsR two-component system might promote conversion of anthranilate to PQS. Taken together, these results establish BqsS-BqsR as a novel two-component system that regulates biofilm decay in P. aeruginosa by modulating biosynthesis of QS signals and rhamnolipids.

Dickeya zeae is the causal agent of rice foot rot and maize stalk rot diseases, which could cause severe economic losses. The pathogen is known to produce two phytotoxins known as zeamine and zeamine II which are also potent antibiotics against both gram-positive and gram-negative bacteria pathogens. Zeamine II is a long-chain aminated polyketide and zeamine shares the same polyketide structure as zeamine II, with an extra valine derivative moiety conjugated to the primary amino group of zeamine II. In this study, we have identified a gene designated as zmsK encoding a putative nonribosomal peptide synthase (NRPS) by screening of the transposon mutants defective in zeamine production. Different from most known NRPS enzymes, which are commonly multidomain proteins, ZmsK contains only a condensation domain. High-performance liquid chromatography and mass spectrometry analyses showed that the ZmsK deletion mutant produced only zeamine II but not zeamine, suggesting that ZmsK catalyzes the amide bond formation by using zeamine II as a substrate to generate zeamine. We also present evidence that a partially conserved catalytic motif within the condensation domain is critical for zeamine production. Furthermore, we show that deletion of zmsK substantially decreased the total antimicrobial activity and virulence of D. zeae. Our findings provide a new insight into the biosynthesis pathway of zeamines and the virulence mechanisms of the bacterial pathogen D. zeae.

The widely used insecticide fenpropathrin in agriculture has become a public concern because of its heavy environmental contamination and toxic effects on mammals, yet little is known about the kinetic and metabolic behaviors of this pesticide. This study reports the degradation kinetics and metabolic pathway of fenpropathrin in Bacillus sp. DG-02, previously isolated from the pyrethroid-manufacturing wastewater treatment system. Up to 93.3% of 50 mg L(-1) fenpropathrin was degraded by Bacillus sp. DG-02 within 72 h, and the degradation rate parameters qmax, Ks, and Ki were determined to be 0.05 h(-1), 9.0 mg L(-1), and 694.8 mg L(-1), respectively. Analysis of the degradation products by gas chromatography-mass spectrometry led to identification of seven metabolites of fenpropathrin, which suggest that fenpropathrin could be degraded first by cleavage of its carboxylester linkage and diaryl bond, followed by degradation of the aromatic ring and subsequent metabolism. In addition to degradation of fenpropathrin, this strain was also found to be capable of degrading a wide range of synthetic pyrethroids including deltamethrin, λ-cyhalothrin, β-cypermethrin, β-cyfluthrin, bifenthrin, and permethrin, which are also widely used insecticides with environmental contamination problems with the degradation process following the first-order kinetic model. Bioaugmentation of fenpropathrin-contaminated soils with strain DG-02 significantly enhanced the disappearance rate of fenpropathrin, and its half-life was sharply reduced in the soils. Taken together, these results depict the biodegradation mechanisms of fenpropathrin and also highlight the promising potentials of Bacillus sp. DG-02 in bioremediation of pyrethroid-contaminated soils.

BackgroundExtensive use of antibiotics has fostered the emergence of superbugs that are resistant to multidrugs, which becomes a great healthcare and public concern. Previous studies showed that quorum sensing signal DSF (diffusible signal factor) not only modulates bacterial antibiotic resistance through intraspecies signaling, but also affects bacterial antibiotic tolerance through interspecies communication. These findings motivate us to exploit the possibility of using DSF and its structurally related molecules as adjuvants to influence antibiotic susceptibility of bacterial pathogens.ResultsIn this study, we have demonstrated that DSF signal and its structurally related molecules could be used to induce bacterial antibiotic susceptibility. Exogenous addition of DSF signal (cis-11-methyl-2-dodecenoic acid) and its structural analogues could significantly increase the antibiotic susceptibility of Bacillus cereus, possibly through reducing drug-resistant activity, biofilm formation and bacterial fitness. The synergistic effect of DSF and its structurally related molecules with antibiotics on B. cereus is dosage-dependent. Combination of DSF with gentamicin showed an obviously synergistic effect on B. cereus pathogenicity in an in vitro model. We also found that DSF could increase the antibiotic susceptibility of other bacterial species, including Bacillus thuringiensis, Staphylococcus aureus, Mycobacterium smegmatis, Neisseria subflava and Pseudomonas aeruginosa.ConclusionThe results indicate a promising potential of using DSF and its structurally related molecules as novel adjuvants to conventional antibiotics for treatment of infectious diseases caused by bacterial pathogens.

Dickeya zeae strain EC1 was recently shown to produce a new type of phytotoxins designated as zeamine and zeamine II, which are potent wide-spectrum antibiotics against Gram-positive and Gram-negative bacterial pathogens, suggesting their promising potential as clinical medicines. In this study, the optimized medium composition and culture conditions for biosynthesis of novel antibiotics zeamines have been established by using response surface methodology, largely increasing the yield of zeamines from original about 7.35 µg·mL−1 in minimal medium to about 150 µg·mL−1 in LS5 medium. The study identified the major factors contributing to zeamines production, which include nitrate, sucrose, asparaginate, mineral elements Mg2+ and K+, and optimized amount of phosphate. In addition, the results showed that overexpression of zmsK in D. zeae strain EC1 could further increase zeamines yield to about 180 µg·mL−1 in LS5 medium. The findings from this study could facilitate further characterization and utilization of these two novel antibiotics, and also provide useful clues for understanding the regulatory mechanisms that govern D. zeae virulence.