Inhibition of Pseudomonas aeruginosa swarming motility by 1-naphthol and other bicyclic compounds bearing hydroxyl groups.

Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols. Pseudomonas aeruginosa, a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants, and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of P. aeruginosa. Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in P. aeruginosa. Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyperswarming P. aeruginosa cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. We showed that this 1-naphthol-dependent swarming inhibition is independent of changes of rhamnolipid production and the intracellular level of signaling molecule cyclic-di-GMP (c-di-GMP). Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and with pyochelin, flagellar, and pilus synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of P. aeruginosa, and these results provide new insight into the chemical structures that inhibit the specific phenotypes of P. aeruginosa.

Effect of abandonment on diversity and abundance of free-living nitrogen-fixing bacteria and total bacteria in the cropland soils of Hulun Buir, Inner Mongolia.

In Inner Mongolia, steppe grasslands face desertification or degradation because of human over activity. One of the reasons for this condition is that croplands have been abandoned after inappropriate agricultural management. The soils in these croplands present heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, we assessed the molecular ecology of total and free-living nitrogen-fixing bacterial communities in soils from steppe grasslands and croplands that were abandoned for different periods (1, 5, and 25 years) and compared the degree of recovery. The abandoned croplands included in the study were natural restoration areas without human activity. Denaturing gradient gel electrophoresis and quantitative PCR (qPCR) were used to analyze the nifH and 16S rRNA genes to study free-living diazotrophs and the total bacterial community, respectively. The diversities of free-living nitrogen fixers and total bacteria were significantly different between each site (P<0.001). Neither the total bacteria nor nifH gene community structure of a cropland abandoned for 25 years was significantly different from those of steppe grasslands. In contrast, results of qPCR analysis of free-living nitrogen fixers and total bacteria showed significantly high abundance levels in steppe grassland (P<0.01 and P<0.03, respectively). In this study, the microbial communities and their gene abundances were assessed in croplands that had been abandoned for different periods. An understanding of how environmental factors and changes in microbial communities affect abandoned croplands could aid in appropriate soil management to optimize the structures of soil microorganisms.

Low concentrations of ethanol stimulate biofilm and pellicle formation in Pseudomonas aeruginosa.

Biofilms are communities of surface-attached microbial cells that resist environmental stresses. In this study, we found that low concentrations of ethanol increase biofilm formation in Pseudomonas aeruginosa PAO1 but not in a mutant of it lacking both Psl and Pel exopolysaccharides. Low concentrations of ethanol also increased pellicle formation at the air-liquid interface.

Membrane vesicle formation is associated with pyocin production under denitrifying conditions in Pseudomonas aeruginosa†PAO1.

Many Gram-negative bacteria produce membrane vesicles (MVs) that serve as vehicles to mediate intraspecies and interspecies interactions. Despite their ubiquity in Gram-negative bacteria and their biological importance, how MV formation is regulated is poorly understood. Pseudomonas aeruginosa is a ubiquitous bacterium that is one of the most extensively studied model organism in MVs. Recent studies highlight the importance of a quorum-sensing signal, Pseudomonas quinolone signal (PQS), in the formation of MVs; however, PQS synthesis requires oxygen and is not produced under anoxic conditions. This situation leads to the question of MV production under anoxic conditions. Here, we examined whether MVs are produced under denitrifying conditions and what kind of factors are involved in the MV production under such condition. Under denitrifying condition, P. aeruginosa PAO1 produced a considerable amount of MVs. Interestingly, pyocin components were found to be accumulated in the isolated MVs. Pyocin-related protein mutants produced less MVs compared with the wild type. We further indicate that pyocin production is activated by nitric oxide, in which the SOS response is involved. This study presents a regulatory mechanism where pyocin is associated with MV production, and further implies how the environment impacts MV production in P. aeruginosa.

Analysis of genes for succinoyl trehalose lipid production and increasing production in Rhodococcus sp. strain SD-74.

Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species.

Three-dimensional visualization of mixed species biofilm formation together with its substratum.

Biofilms, such as dental plaque, are aggregates of microorganisms attached to a surface. Thus, visualization of biofilms together with their attached substrata is important in order to understand details of the interaction between them. However, so far there is limited availability of such techniques. Here, non-invasive visualization of biofilm formation with its attached substratum by applying the previously reported technique of continuous-optimizing confocal reflection microscopy (COCRM) is reported. The process of development of oral biofilm together with its substratum was sequentially visualized with COCRM. This study describes a convenient method for visualizing biofilm and its attached surface.

Dechlorination of chloral hydrate is influenced by the biofilm adhesin protein LapA in Pseudomonas putida LF54.

LapA is the largest surface adhesion protein of Pseudomonas putida that initiates biofilm formation. Here, by using transposon insertion mutagenesis and a conditional lapA mutant, we demonstrate for the first time that LapA influences chloral hydrate (CH) dechlorination in P. putida LF54.

Interspecies interaction between Pseudomonas aeruginosa and other microorganisms.

Microbes interact with each other in multicellular communities and this interaction enables certain microorganisms to survive in various environments. Pseudomonas aeruginosa is a highly adaptable bacterium that ubiquitously inhabits diverse environments including soil, marine habitats, plants and animals. Behind this adaptivity, P. aeruginosa has abilities not only to outcompete others but also to communicate with each other to develop a multispecies community. In this review, we focus on how P. aeruginosa interacts with other microorganisms. P. aeruginosa secretes antimicrobial chemicals to compete and signal molecules to cooperate with other organisms. In other cases, it directly conveys antimicrobial enzymes to other bacteria using the Type VI secretion system (T6SS) or membrane vesicles (MVs). Quorum sensing is a central regulatory system used to exert their ability including antimicrobial effects and cooperation with other microbes. At least three quorum sensing systems are found in P. aeruginosa, Las, Rhl and Pseudomonas quinolone signal (PQS) systems. These quorum-sensing systems control the synthesis of extracellular antimicrobial chemicals as well as interaction with other organisms via T6SS or MVs. In addition, we explain the potential of microbial interaction analysis using several micro devices, which would bring fresh sensitivity to the study of interspecies interaction between P. aeruginosa and other organisms.

Cultivation-independent identification of candidate dehalorespiring bacteria in tetrachloroethylene degradation.

Tetrachloroethylene (PCE) is one of the major pollutants and is degraded by dissimilation by dehalorespiring bacteria. The dehalorespiring bacteria are anaerobic, and most cannot be cultured by conventional agar plating methods. Therefore, to identify the dehalorespiring bacteria that dissimilatively degrade PCE, a cultivation-independent method is required. To achieve accurate and detailed analysis of the bacteria, we developed a novel stable isotope probing (SIP) method. This technique involves 2 steps, namely, a labeling step, in which a labeled carbon source is incorporated into the sample's DNA, and an analysis step, in which the DNA is isolated, fractionated, and analyzed by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Subsequently, 16S rRNA sequencing and phylogenetic analysis were performed to identify the bacteria. Initially, we examined the effectiveness of this method by using Dehalococcoides ethenogenes 195 consortium as a defined model system. The result indicated the method was able to correctly identify the dehalorespiring bacteria D. ethenogenes 195 from the consortium. Moreover, in an artificially contaminated microcosm experiment, we confirmed that the method was able to identify the indigenous dehalorespiring bacteria Dehalobacter sp. Thus, we concluded that this novel method was a feasible tool to identify dehalorespiring bacteria in natural environments.

Social Behaviours under Anaerobic Conditions in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is well adapted to grow in anaerobic environments in the presence of nitrogen oxides by generating energy through denitrification. Environmental cues, such as oxygen and nitrogen oxide concentrations, are important in regulating the gene expression involved in this process. Recent data indicate that P. aeruginosa also employs cell-to-cell communication signals to control the denitrifying activity. The regulation of denitrification by these signalling molecules may control nitric oxide production. Nitric oxide, in turn, functions as a signalling molecule by activating certain regulatory proteins. Moreover, under denitrifying conditions, drastic changes in cell physiology and cell morphology are induced that significantly impact group behaviours, such as biofilm formation.

Case study of the relationship between fungi and bacteria associated with high-molecular-weight polycyclic aromatic hydrocarbon degradation.

Although bacteria play dominant roles in microbial bioremediation, few of them have been reported that were capable of utilizing high-molecular-weight (HMW) organic pollutants as their sole sources of carbon and energy. However, many soil fungi can metabolize those of pollutants, although they rarely complete mineralization. In this paper, we investigated the dynamic relationship between fungi and bacteria associated with degradation of HMW-polycyclic aromatic hydrocarbons (PAHs). Artificial fungal-bacterial mixed cultures were constructed to simulate the environment of actual polluted sites. Four bacterial strains and seven fungal strains were isolated that related to the removal of phenanthrene, fluoranthene and pyrene in the soil. Furthermore, these strains were used to create mixed culture of bacteria (Bact-mix), mixed culture of fungi (Fung-mix), fungal-bacterial co-cultures (Fung-Bact), respectively. The maximal pyrene removal rate (67%, 28days) was observed in the Fung-Bact, compared with cultures of Fung-mix (39%) and Bact-mix (56%). The same tendency was also indicated in the degradation of phenanthrene and fluoranthene. In addition, a dynamic relationship during the degradation process between fungi and bacteria was monitored through using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method.

Multifunctional membrane vesicles in Pseudomonas aeruginosa.

Gram-negative bacteria secrete small particles called membrane vesicles (MVs) into the extracellular milieu. While MVs have important roles in delivering toxins from pathogenic bacteria to eukaryotic cells, these vesicles also play ecological roles necessary for survival in various environmental conditions. Pseudomonas aeruginosa, which lives in soil, ocean, plant, animal and human environments, has become a model organism for studying these small extracellular particles. Such studies have increased our understanding of the function and biogenesis of bacterial MVs. Pseudomonas aeruginosa MVs possess versatile components and chemical substances with unique structures. These characteristics allow MVs to play their multifunctional biological roles, including microbial interaction, maintenance of biofilm structure and host infection. This review summarizes the comprehensive biochemical and physiochemical properties of MVs derived from P. aeruginosa. These studies will help us understand their biological roles of MVs not only in pathogenicity but also in microbial ecology. Also, the mechanisms of MV production, as currently understood, are discussed.

Characterization of phospholipids in membrane vesicles derived from Pseudomonas aeruginosa.

Many Gram-negative bacteria release membrane vesicles (MVs), but their phospholipid properties are poorly understood. Phosphatidylglycerol was present at high levels in MVs derived from Pseudomonas aeruginosa, but not in the cellular outer membrane. The ratio of stearic acid in MVs was high compared to that in the cellular outer membrane. These findings suggest that membrane rigidity is associated with MV biogenesis.

Assimilative and co-metabolic degradation of chloral hydrate by bacteria and their bioremediation potential.

Although the bacterial degradation of chloral hydrate (CH) has been recognized for several decades, its degradation pathway by assimilation has not been demonstrated. In this paper, we report the isolation of the LF54 bacterial strain, which utilizes CH as its sole carbon and energy source. LF54 converted CH into trichloroethanol (TCAol), which was dehalogenated to dichloroethanol (DCAol), and CO(2) was detected as the end product. Another strain that we isolated, RS20, co-metabolized CH into TCAol. Our 16S rRNA gene sequencing and taxonomic analyses revealed that the LF54 and RS20 strains belong to the Pseudomonas and Arthrobacter genera, respectively. When the two strains were inoculated into soil microcosms, both degraded 0.3mM CH to undetectable levels (<0.01mM) within 5days. These results suggest that LF54 and RS20 could be used in the bioremediation of CH-contaminated environments.

Monitoring biofilm development in a microfluidic device using modified confocal reflection microscopy.

The feasibility of a method to monitor biofilm development non-destructively in a microfluidic device was addressed. Here, we report that biofilm growth could be non-destructively monitored by an image analysis technique based on modification of confocal reflection microscopy.

Bacterial growth monitoring in a microfluidic device by confocal reflection microscopy.

The feasibility of a method to nondestructively measure planktonic bacterial growth in a microfluidic device was addressed. Here, we report that the growth of Pseudomonas aeruginosa in a microfluidic device could be measured by a three-dimensional image analysis technique based on confocal reflection microscopy in a time-course.

Variation of physiochemical properties and cell association activity of membrane vesicles with growth phase in Pseudomonas aeruginosa.

Pseudomonas aeruginosa and other Gram-negative bacteria release membrane vesicles (MVs) from their surfaces, and MVs have an ability to interact with bacterial cells. Although it has been known that many bacteria have mechanisms that control their phenotypes with the transition from exponential phase to stationary phase, changes of properties in released MVs have been poorly understood. Here, we demonstrate that MVs released by P. aeruginosa during the exponential and stationary phases possess different physiochemical properties. MVs purified from the stationary phase had higher buoyant densities than did those purified from the exponential phase. Surface charge, characterized by zeta potential, of MVs tended to be more negative as the growth shifted to the stationary phase, although the charges of PAO1 cells were not altered. Pseudomonas quinolone signal (PQS), one of the regulators related to MV production in P. aeruginosa, was lower in MVs purified from the exponential phase than in those from the stationary phase. MVs from the stationary phase more strongly associated with P. aeruginosa cells than did those from the exponential phase. Our findings suggest that properties of MVs are altered to readily interact with bacterial cells along with the growth transition in P. aeruginosa.

Bicyclic compounds repress membrane vesicle production and Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa secretes membrane vesicles (MVs) that deliver several virulence factors as a cargo. We found that indole and its derivative compounds, including 4-hydroxyindole, 5-hydroxyindole, 6-hydroxyindole and isatin, repress MV production significantly. These compounds also repressed the synthesis of Pseudomonas quinolone signal (PQS), which is one of the quorum-sensing signals that upregulate virulence gene expression and positively control MV production. Moreover, we showed that other bicyclic compounds, including 1-naphthol, 2-naphthol, 2,3-dihydroxynaphthalene, 1-aminonaphthalene and 8-quinolinol, significantly repress MV production and PQS synthesis. In conclusion, we provide new information about the chemical structures that inhibit P. aeruginosa virulence.

The effect of a cell-to-cell communication molecule, Pseudomonas quinolone signal (PQS), produced by P. aeruginosa on other bacterial species.

One of the most important factors in the development of a bacterial community is whether the bacteria are able to grow in that habitat. The regulation of bacterial growth is generally studied in relation to physicochemical conditions, however, how bacterial communities regulate themselves remains unclear. In our previous study, it was demonstrated that a cell-to-cell communication molecule, 2-heptyl-3-hydroxy-4-quinolone, referred to as the Pseudomonas quinolone signal (PQS), affects respiring-activity in Pseudomonas aeruginosa without requiring its cognate receptor PqsR. The results suggested that PQS may affect other bacterial species, which was further examined in this study. PQS repressed the growth of several species including both Gram-negative and Gram-positive bacteria. In most cases, this effect differed from the bacteriostatic or bacteriolytic actions of antibiotics. The growth repression by PQS was inhibited when iron was added to the medium, indicating iron-chelating activity to be involved. In addition, PQS affected oxygen consumption in some species tested, and may have other underlying effects. Thus, this cell-to-cell communication molecule may influence the development of bacterial communities by regulating bacterial growth, and physicochemical factors such as iron would be important in determining its effect.

Visualizing the effects of biofilm structures on the influx of fluorescent material using combined confocal reflection and fluorescent microscopy.

Mass transport into the interior of biofilms and biological aggregates is a critical factor that affects their metabolic activity. In this study, we demonstrated the utility of a simple procedure that combines confocal reflection microscopy and fluorescent confocal laser scanning microscopy to visually explain the effects of biofilm structure on mass transport.