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.

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.

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.

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.

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.

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.

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.

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.

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.

Outer membrane machinery and alginate synthesis regulators control membrane vesicle production in Pseudomonas aeruginosa.

The opportunistic human bacterial pathogen Pseudomonas aeruginosa produces membrane vesicles (MVs) in its surrounding environment. Several features of the P. aeruginosa MV production mechanism are still unknown. We previously observed that depletion of Opr86, which has a role in outer membrane protein (OMP) assembly, resulted in hypervesiculation. In this study, we showed that the outer membrane machinery and alginate synthesis regulatory machinery are closely related to MV production in P. aeruginosa. Depletion of Opr86 resulted in increased expression of the periplasmic serine protease MucD, suggesting that the accumulation of misfolded OMPs in the periplasm is related to MV production. Indeed, the mucD mutant showed a mucoid phenotype and the mucD mutation caused increased MV production. Strains with the gene encoding alginate synthetic regulator AlgU, MucA, or MucB deleted also caused altered MV production. Overexpression of either MucD or AlgW serine proteases resulted in decreased MV production, suggesting that proteases localized in the periplasm repress MV production in P. aeruginosa. Deletion of mucD resulted in increased MV proteins, even in strains with mutations in the Pseudomonas quinolone signal (PQS), which serves as a positive regulator of MV production. This study suggests that misfolded OMPs may be important for MV production, in addition to PQS, and that these regulators act in independent pathways.

Stress resistance of biofilm and planktonic Lactobacillus plantarum subsp. plantarum JCM 1149.

The purpose of this study was to investigate the change in resistance of biofilm and planktonic food spoilage lactic acid bacteria (LAB) to environmental stresses, which strongly inhibit bacterial growth and are important in food preservation or in disinfection. The stress responses of biofilm and planktonic cells of Lactobacillus plantarum subsp. plantarum JCM 1149, which was used as a model spoilage bacterium, in various organic acids (namely, acetic acid, citric acid, lactic acid, and malic acid), ethanol, and sodium hypochlorite, were investigated using survival tests. The bacterial cells in biofilms showed greater resistance to all treatments than the planktonic bacterial cells in either the stationary or logarithmic phase. The planktonic bacterial cells showed reduced resistance to acetic acid after the cell suspension was diluted; however, intriguingly, the bacterial cells in biofilms maintained their resistance to acetic acid even after they were suspended or the cell suspension was diluted. These findings suggested the risk for food spoilage due to LAB derived from biofilms and suspended or diluted in foods, and demonstrated the importance of controlling biofilms of LAB in the food industry.

Influence of the Pseudomonas quinolone signal on denitrification in Pseudomonas aeruginosa.

Denitrification is a well-studied respiratory system that is also important in the biogeochemical nitrogen cycle. Environmental signals such as oxygen and N-oxides have been demonstrated to regulate denitrification, though how denitrification is regulated in a bacterial community remains obscure. Pseudomonas aeruginosa is a ubiquitous bacterium that controls numerous genes through cell-to-cell signals. The bacterium possesses at least two N-acyl-L-homoserine lactone (AHL) signals. In our previous study, these quorum-sensing signals controlled denitrification in P. aeruginosa. In addition to the AHL signals, a third cell-to-cell communication signal, 2-heptyl-3-hydroxy-4-quinolone, referred to as the Pseudomonas quinolone signal (PQS), has been characterized. In this study, we examined the effect of PQS on denitrification to obtain more insight into the respiratory regulation in a bacterial community. Denitrification in P. aeruginosa was repressed by PQS, which was partially mediated by PqsR and PqsE. Measuring the denitrifying enzyme activities indicated that nitrite reductase activity was increased by PQS, whereas PQS inhibited nitric oxide reductase and the nitrate-respiratory chain activities. This is the first report to demonstrate that PQS influences enzyme activities, suggesting this effect is not specific to P. aeruginosa. Furthermore, when iron was supplied to the PQS-added medium, denitrifying activity was almost restored, indicating that the iron chelating property of PQS affected denitrification. Thus, our data indicate that PQS regulates denitrification primarily through iron chelation. The PQS effect on denitrification was relevant in a condition where oxygen was limited and denitrification was induced, suggesting its role in controlling denitrification where oxygen is present.

Opr86 is essential for viability and is a potential candidate for a protective antigen against biofilm formation by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is one of the most refractory to therapy when it forms biofilms in the airways of cystic fibrosis patients. To date, studies regarding the production of an immunogenic and protective antigen to inhibit biofilm formation by P. aeruginosa have been superficial. The previously uncharacterized outer membrane protein (OMP) Opr86 (PA3648) of P. aeruginosa is a member of the Omp85 family, of which homologs have been found in all gram-negative bacteria. Here we verify the availability of Opr86 as a protective antigen to inhibit biofilm formation by P. aeruginosa PAO1 and several other isolates. A mutant was constructed in which Opr86 expression could be switched on or off through a tac promoter-controlled opr86 gene. The result, consistent with previous Omp85 studies, showed that Opr86 is essential for viability and plays a role in OMP assembly. Depletion of Opr86 resulted in streptococci-like morphological changes and liberation of excess membrane vesicles. A polyclonal antibody against Opr86 which showed reactivity to PAO1 cells was obtained. The antibody inhibited biofilm formation by PAO1 and the other clinical strains tested. Closer examination of early attachment revealed that cells treated with the antibody were unable to attach to the surface. Our data suggest that Opr86 is a critical OMP and a potential candidate as a protective antigen against biofilm formation by P. aeruginosa.

Development of a novel biofilm continuous culture method for simultaneous assessment of architecture and gaseous metabolite production.

The way that gaseous metabolite production changes along with biofilm architecture development is poorly understood. To address this question, we developed a novel flow reactor biofilm culture method that allows for simultaneous assessment of gaseous metabolite production and architecture visualization. In this report, we establish the utility of this method using denitrification by Pseudomonas aeruginosa biofilms as a model system. Using this method, we were able to collect and analyze gaseous metabolites produced by denitrification and also visualize biofilm architecture in a nondestructive manner. Thus, we propose that this novel method is a powerful tool to investigate potential relationships between biofilm architecture and the gas-producing metabolic activity of biofilms, providing new insights into biofilm ecology.

Diversity and similarity of microbial communities in petroleum crude oils produced in Asia.

To understand microbial communities in petroleum crude oils, we precipitated DNA using high concentrations of 2,2,4-trimethylpentane (isooctane) and purified. Samples of DNA from five crude oils, (Middle East, 3; China, 1; and Japan, 1) were characterized based upon their 16S rRNA gene sequences after PCR amplification and the construction of clone libraries. We detected 48 eubacterial species, one cyanobacterium, and one archaeon in total. The microbial constituents were diverse in the DNA samples. Most of the bacteria affiliated with the sequences of the three oils from the Middle East comprised similar mesophilic species. Acinetobacter, Propionibacterium, Sphingobium and a Bacillales were common. In contrast, the bacterial communities in Japanese and Chinese samples were unique. Thermophilic Petrotoga-like bacteria (11%) and several anaerobic-thermophilic Clostridia- and Synergistetes-like bacteria (20%) were detected in the Chinese sample. Different thermophiles (12%) and Clostridia (2%) were detected in the Japanese sample.

Reduction of chlortetracycline-resistant Escherichia coli in weaned piglets fed fermented liquid feed.

We investigated the change in chlortetracycline resistance in 360 Escherichia coli strains separated from the feces of piglets fed fermented liquid feed (FLF) in comparison with those fed dry feed (control). The total amount of lactic acid bacteria in feces was 8.77 x 10(8) CFU/g DM at weaning, which increased to 1.23 x 10(12) CFU/g DM (FLF) at 28 days after weaning (P<0.001). As a result of the antibiotic susceptibility test, almost all isolates were chlortetracycline-resistant (CTCR) until 14 days after weaning both in the FLF and control groups. At 28 days, the CTCR E. coli decreased to 22.2% in the FLF group, while the proportion of resistant bacteria was 88.9% in the control group. In addition, as a result of the gene analysis, it was clarified that there is a relation between the decrease in the minimum inhibitory concentration values and the possession rate of the tetracycline-resistance gene tet(A), tet(B) and tet(D). These results show that FLF caused an increase in the number of lactic acid bacteria in the intestines, and suggested that the feeding of FLF can possibly reduce antibiotic-resistance bacteria.

Identification and functional analysis of genes required for desulfurization of alkyl dibenzothiophenes of Mycobacterium sp. G3.

Mycobacterium sp. G3 was reported as a dibenzothiophene (DBT)-degrading microorganism and the first strain to have the ability to degrade high-molecular-weight alkyl DBTs, such as 4,6-dibutyl DBT and 4,6-dipentyl DBT, by the C-S bond cleavage pathway. Three genes (mdsA, mdsB, and mdsC) for desulfurization, which form a cluster, were cloned from Mycobacterium sp. G3. The expression of each gene in Escherichia coli JM109 showed that MdsC oxidized DBT to DBT sulfone, MdsA transformed DBT sulfone into 2-(2'-hydroxyphenyl)benzene sulfinate (HPBS), and MdsB desulfinated HPBS into 2-hydroxybiphenyl (HBP), indicating that the gene products of mdsABC are functional in the recombinant. MdsC oxidized 4,6-dimethyl DBT, 4,6-diethyl DBT, 4,6-dipropyl DBT and 4,6-dibutyl DBT to each sulfone form, suggesting that MdsC covers a broad specificity for alkyl DBTs.

Screening of novel cellulose-degrading bacterium and its application to denitrification of groundwater.

To establish an environmentally friendly groundwater bioremediation process using a cellulose carrier combined with cellulose-utilizing, denitrifying microorganisms, a novel psychrophilic bacterium, designated CL-5, which can degrade a commercial-based cellulose carrier as the sole carbon source, was screened. Since the denitrification capability of CL-5 is low, complex microbial systems were constructed together with other denitrifying bacteria designated NR-1 and NR-2 that were also isolated from soil. The nitrate-reducing activities of mixed cultures were much higher than those of the pure cultures of CL-5, NR-1 and NR-2. The highest N(2)O and N(2) formation activities were observed in the mixed culture of CL-5+NR-2.

Matrix metalloproteinase-2 inhibition and Zn2+-chelating activities of pyoverdine-type siderophores.

Pyoverdine-type siderophores from fluorescent Pseudomonas species were purified by Zn2+-chelate chromatography, and their matrix metalloproteinase-2 (MMP-2) inhibition and metal-ion-chelating activities were studied. Structurally different pyoverdines showed different MMP-2 inhibition activities, and the inhibition activity was correlated with Zn2+-chelating activity. The IC50 value of a pyoverdine ((P113A1)-2, MW 1187) for MMP-2 was 0.27 microg/ml (0.23 microM).