Hideaki Maseda

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

ABSTRACT nfxC-type cells of Pseudomonas aeruginosa that produce the MexEF-OprN efflux pump exhibit resistance to fluoroquinolones and chloramphenicol and hypersusceptibility to most classical β-lactam antibiotics. We investigated the molecular mechanism of how the nfxC mutation causes β-lactam hypersusceptibility. The MexAB-OprM extrusion pump transports and confers resistance to β-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 β-lactam hypersusceptibility in nfxC-type mutant cells is caused by MexT-mediated cancellation of C4-HSL-mediated enhancement of MexAB-OprM expression.

Assignment of the Substrate-Selective Subunits of the MexEF-OprN Multidrug Efflux Pump of Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa expresses a low level of the MexAB-OprM efflux pump and shows natural resistance to many structurally and functionally diverse antibiotics. The mutation that has been referred to previously as nfxC expresses an additional efflux pump, MexEF-OprN, exhibiting resistance to fluoroquinolones, imipenem, and chloramphenicol and hypersusceptibility to β-lactam antibiotics. To address the antibiotic specificity of the MexEF-OprN efflux pump, we introduced a plasmid carrying themexEF-oprN operon into P. aeruginosa lacking the mexAB-oprM operon. The transformants exhibited resistance to fluoroquinolones, trimethoprim, and chloramphenicol but, unlike most nfxC-type mutants, did not show β-lactam hypersusceptibility. The transformants exhibited additional resistance to tetracycline. In the next experiment, we analyzed the MexEF-OprN pump subunit(s) responsible for substrate selectivity by expressing MexE, MexF, OprN, and MexEF in strains lacking MexA, MexB, OprM, and MexAB, respectively. The MexEF-OprM/ΔMexAB transformants exhibited MexEF-OprN-type pump function that rendered the strains resistant to fluoroquinolones and chloramphenicol but did not change susceptibility to β-lactam antibiotics compared with the host strain. The MexAB-OprN/ΔOprM, MexAF-OprM/ΔMexB, and MexEB-OprM/ΔMexA mutants exhibited antibiotic susceptibility indistinguishable from that in the mutant lacking both types of efflux pumps. The results imply that the MexEF-OprM pump selects substrates by a MexEF functional unit. Interestingly, OprN did not link functionally with the MexAB complex, despite the fact that OprM interacted functionally with MexEF.

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.

Mutational Upregulation of a Resistance-Nodulation-Cell Division-Type Multidrug Efflux Pump, SdeAB, upon Exposure to a Biocide, Cetylpyridinium Chloride, and Antibiotic Resistance in Serratia marcescens

ABSTRACT Serratia marcescens is an important opportunistic pathogen in hospitals, where quaternary ammonium compounds are often used for disinfection. The aim of this study is to elucidate the effect of a biocide on the emergence of biocide- and antibiotic-resistant mutants and to characterize the molecular mechanism of biocide resistance in Serratia marcescens. A quaternary ammonium compound-resistant strain, CRes01, was selected by exposing a wild-type strain of S. marcescens to cetylpyridinium chloride. The CRes01 cells exhibited 2- to 16-fold more resistance than the wild-type cells to biocides and antibiotics, including cetylpyridinium chloride, benzalkonium chloride, chlorhexidine gluconate, fluoroquinolones, tetracycline, and chloramphenicol, and showed increased susceptibilities to β-lactam antibiotics and N-dodecylpyridinium iodide. Mutant cells accumulated lower levels of norfloxacin than the parent cells in an energized state but not in a de-energized state, suggesting that the strain produced a multidrug efflux pump(s). To verify this assumption, we knocked out a putative efflux pump gene, sdeAB, in CRes01 and found that the knockout restored susceptibility to most quaternary ammonium compounds and antibiotics, to which the CRes01 strain showed resistance. On the basis of these and other results, we concluded that S. marcescens gains resistance to both biocides and antibiotics by expressing the SdeAB efflux pump upon exposure to cetylpyridinium chloride.

Characteristics of a Microcystin-Degrading Bacterium under Alkaline Environmental Conditions

The pH of the water associated with toxic blooms of cyanobacteria is typically in the alkaline range; however, previously only microcystin-degrading bacteria growing in neutral pH conditions have been isolated. Therefore, we sought to isolate and characterize an alkali-tolerant microcystin-degrading bacterium from a water bloom using microcystin-LR. Analysis of the 16S rRNA gene sequence revealed that the isolated bacterium belonged to the genus Sphingopyxis, and the strain was named C-1. Sphingopyxis sp. C-1 can grow; at pH 11.0; however, the optimum pH for growth was pH 7.0. The microcystin degradation activity of the bacterium was the greatest between pH 6.52 and pH 8.45 but was also detected at pH 10.0. The mlrA homolog encoding the microcystin-degrading enzyme in the C-1 strain was conserved. We concluded that alkali-tolerant microcystin-degrading bacterium played a key role in triggering the rapid degradation of microcystin, leading to the disappearance of toxic water blooms in aquatic environments.

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.

Enzymatic pathway for biodegrading microcystin LR in Sphingopyxis sp. C-1

The mlr gene cluster consisting of mlrA, mlrB, mlrC, and mlrD is involved in the degradation of the cyanobacterial toxin microcystin. However, it is unclear which degradation intermediates are metabolized by MlrB and MlrC. To address these questions, we constructed recombinant Escherichia coli to overproduce MlrB and MlrC from Sphingopyxis sp. C-1, and determined which intermediates were degraded in cell-free extracts. The cell-free extract containing MlrB degraded linearized microcystin-LR, giving rise to a tetrapeptide. The cell-free extract of MlrC degraded linearized microcystin-LR and also degraded the tetrapeptide to the amino acid Adda. These results indicate that linearized microcystin-LR is degraded by both MlrB and MlrC, and tetrapeptide is degraded by specifically by MlrC in Sphingopyxis sp. C-1.

Investigations into the biodegradation of microcystin-LR mediated by the biofilm in wintertime from a biological treatment facility in a drinking-water treatment plant

The potential of winter biofilm for microcystin-LR (MCLR) biodegradation was comparatively evaluated under various nutrient conditions. Results indicated that MCLR was completely biodegraded by Day 7 without nutrient addition. MCLR-biodegradation was inhibited in the presence of phosphate or glucose addition, with complete MCLR removal observed by Day 10. MCLR was totally biodegraded by Day 7 with dual nutrients comprising glucose and nitrate, suggesting that additional nitrate alleviated the inhibitory effect of glucose alone on the biodegradation. Simultaneously, MCLR-degrading gene (mlrA) abundance were detected to increase with increasing amount of MCLR being degraded under the respective conditions, implying that MCLR-biodegradation depended on the population of indigenous MCLR-degrading bacteria (MCLRDB), which was related to the population of non-degrading bacteria in the biofilm. MCLRDB was found to primarily use MCLR for proliferation rather than other nutrients. This is the first report verifying MCLR as a primary substrate for bacteria under various nutrient conditions.

Plasmid marker rescue transformation in Thermus thermophilus

Abstract The marker rescue transformation method was examined for Thermus thermophilus . Plasmid pYK134, constructed from a Thermus cryptic plasmid pTT8 with a modified staphylococcal kanamycin resistance (Km r ) gene and a heterologous Thermus trpB gene as selection markers was used to transform T. thermophilus HB27 trpB mutant. The transformation efficiency with pYK134 against a plasmidless recipient was about 1 × 107 per microgram of DNA, or about 0.1% per viable cell count for both Km r and Trp + markers. When the pTT8-carrying HB27 trpB mutant was used as a recipient, transformation efficiency with pYK134 increased about 30-fold, i.e. about 3 x 108 per microgram of DNA or 3% per viable cell count. The plasmidless recipient could not be transformed with linearized pYK134, whereas the pTT8-carrying recipient was transformed by it only when linearization was done at the pTT8 region in pYK134. These results suggest that flanking homologous regions are necessary for plasmid marker rescue transformation in T. thermophilus .

Development of expression vectors for Thermus thermophilus

We constructed expression vectors for Thermus thermophilus. The transformation efficiency of pTEV131 per viable cells was 0.15% at 60°C and 0.11% at 68°C, and with marker rescue transformation, it was 1.9% at 60°C and 0.5% at 68°C. When the crtB gene was inserted into the cloning sites of this vector, the transformant expressed 3-fold more crtB gene than the wild type.