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.

Production and degradation of N-acylhomoserine lactone quorum sensing signal molecules in bacteria isolated from activated sludge.

N-Acylhomoserine lactones (AHLs) function as quorum-sensing signaling molecules in many Gram-negative bacteria. We isolated a total of 672 bacterial strains from activated sludge obtained from seven sewage treatment plants in Tochigi Prefecture, Japan, and screened for AHL-producing and degrading strains. Isolates (n = 107) stimulated AHL-mediated purple pigment production in AHL reporter strains Chromobacterium violaceum CV026 and VIR07. Based on their 16S rRNA gene sequences, most of these AHL-producing isolates were assigned to the genus Aeromonas, and they were divided into six groups. Isolates (n = 46) degraded N-decanoyl-L-homoserine lactone (C10-HSL) within 24 h. Based on their 16S rRNA gene sequences, the most dominant AHL-degrading isolates were assigned to the genus Acinetobacter and divided into six groups. Strains Ooi24, Omo91, and Uzu81, which showed higher C10-HSL-degrading activity, showed putative AHL-acylase activity.

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.

Dimerization of the ATRIP protein through the coiled-coil motif and its implication to the maintenance of stalled replication forks.

ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.

Wave-number dependent current correlation for a harmonic oscillator.

The wave-number k dependent current-correlation function is considered for a harmonic oscillator model. An explicit analytic expression for the Laplace transformed correlation function is derived. It is compared with numerical solutions and results obtained by the recurrence relation method. Several limiting cases such as the long-wavelength limit k→0 and the deep inelastic limit k→∞ are discussed in detail. In particular, we show that the deep inelastic limit allows for an explicit summation of the continued fraction. An approximation scheme for the recurrants at intermediate values of k is also considered.

The Response of Pseudomonas aeruginosa PAO1 Efflux Pump-Defective Mutants to N-Octanoyl-L-Homoserine Lactone.

N-Octanoyl-L-homoserine lactone (C8-HSL) is an acyl-homoserine-lactone signal utilized in the quorum-sensing (QS) systems of Burkholderia cenocepacia and other bacterial species. Although also produced by Pseudomonas aeruginosa, its role in this species has not been elucidated. Here, we report that C8-HSL modulated antibiotic resistance and pyocyanin production in a P. aeruginosa efflux pump-deficient mutant. The rhl/las quorum-sensing system and qscR gene were both shown to be nonessential in the C8-HSL-induced changes in ofloxacin resistance, suggesting that P. aeruginosa possesses a distinct pathway to respond to C8-HSL.

Quorum sensing regulates denitrification in Pseudomonas aeruginosa PAO1.

Anaerobic growth of Pseudomonas aeruginosa PAO1 was affected by quorum sensing. Deletion of genes that produce N-acyl-l-homoserine lactone signals resulted in an increase in denitrification activity, which was repressed by exogenous signal molecules. The effect of the las quorum-sensing system was dependent on the rhl quorum-sensing system in regulating denitrification.

Enhancement of the mexAB-oprM efflux pump expression by a quorum-sensing autoinducer and its cancellation by a regulator, MexT, of the mexEF-oprN efflux pump operon in Pseudomonas aeruginosa.

nfxC-type cells of Pseudomonas aeruginosa that produce the MexEF-OprN efflux pump exhibit resistance to fluoroquinolones and chloramphenicol and hypersusceptibility to most classical beta-lactam antibiotics. We investigated the molecular mechanism of how the nfxC mutation causes beta-lactam hypersusceptibility. The MexAB-OprM extrusion pump transports and confers resistance to beta-lactam antibiotics. Interestingly, expression of the mexAB-oprM operon reached the highest level during the mid-stationary growth phase in both wild-type and nfxC-type mutant strains, suggesting that expression of the mexAB-oprM operon may be controlled by cell density-dependent regulation such as quorum sensing. This assumption was verified by demonstrating that exogenous addition of the quorum-sensing autoinducer N-butyryl-L-homoserine lactone (C4-HSL) enhanced the expression of MexAB-OprM, whereas N-(3-oxododecanoyl)-L-homoserine lactone had only a slight effect. Furthermore, this C4-HSL-mediated enhancement of mexAB-oprM expression was repressed by MexT, a positive regulator of the mexEF-oprN operon. It was concluded that beta-lactam hypersusceptibility in nfxC-type mutant cells is caused by MexT-mediated cancellation of C4-HSL-mediated enhancement of MexAB-OprM expression.

A quorum-sensing autoinducer enhances the mexAB-oprM efflux-pump expression without the MexR-mediated regulation in Pseudomonas aeruginosa.

We have previously described that the quorum-sensing autoinducer, N-butyryl-L-homoserine lactone (C4-HSL) enhances mexAB-oprM expression, and this C4-HSL-mediated enhancement of mexAB-oprM expression was repressed by MexT, a positive regulator of the mexEF-oprN operon. In this study, we investigated the interaction between C4-HSL and mexR by using a knockout mutant. It was indicated that the C4-HSL-mediated enhancement of mexAB-oprM expression occurred without MexR-mediated regulation. Furthermore, it was observed that the C4-HSL-mediated enhancement of mexAB-oprM expression without the MexR-mediated regulation was repressed by MexT.