Isolation and Characterization of a 4-Nitrotoluene-Oxidizing Enzyme from Activated Sludge by a Metagenomic Approach

Kimura, Nobutada; Sakai, Kayoko; Nakamura, Kazunori

doi:10.1264/jsme2.me10110

Cited by 20 publications

(19 citation statements)

References 49 publications

(37 reference statements)

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“…Fosmid libraries from sludge and soil were constructed using the CopyControl fosmid library production kit (Epicentre) according to the manufacturer's protocols. The transformants were selected on LB agar plates containing 12.5 g ml Ϫ1 chloramphenicol (LB/Cm) (22). Recombinant fosmids and polymorphisms of the insert DNA were examined by agarose gel electrophoresis of EcoRIdigested purified fosmids from randomly selected E. coli transformants.…”

Section: Methodsmentioning

confidence: 99%

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Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach

Nasuno

Kimura

Fujita

et al. 2012

Appl Environ Microbiol

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A great deal of research has been done to understand bacterial cell-to-cell signaling systems, but there is still a large gap in our current knowledge because the majority of microorganisms in natural environments do not have cultivated representatives. Metagenomics is one approach to identify novel quorum sensing (QS) systems from uncultured bacteria in environmental samples. In this study, fosmid metagenomic libraries were constructed from a forest soil and an activated sludge from a coke plant, and the target genes were detected using a green fluorescent protein (GFP)-based Escherichia coli biosensor strain whose fluorescence was screened by spectrophotometry. DNA sequence analysis revealed two pairs of new LuxI family N-acyl-L-homoserine lactone (AHL) synthases and LuxR family transcriptional regulators (clones N16 and N52, designated AubI/AubR and AusI/ AusR, respectively). AubI and AusI each produced an identical AHL, N-dodecanoyl-L-homoserine lactone (C 12 -HSL), as determined by nuclear magnetic resonance (NMR) and mass spectrometry. Phylogenetic analysis based on amino acid sequences suggested that AusI/AusR was from an uncultured member of the Betaproteobacteria and AubI/AubR was very deeply branched from previously described LuxI/LuxR homologues in isolates of the Proteobacteria. The phylogenetic position of AubI/AubR indicates that they represent a QS system not acquired recently from the Proteobacteria by horizontal gene transfer but share a more ancient ancestry. We demonstrated that metagenomic screening is useful to provide further insight into the phylogenetic diversity of bacterial QS systems by describing two new LuxI/LuxR-type QS systems from uncultured bacteria. Bacteria interact with one another using chemical molecules as sensing signals. Detection of the molecules allows bacteria to distinguish between low and high cell population densities and to control gene expression in response to changes in cell number (46) and local environment (12). This process, referred to as quorum sensing (QS), allows a population of bacteria to coordinately control gene expression. Several types of QS signals have been found, including N-acyl-L-homoserine lactone (AHL) in the Proteobacteria (5,45,46). AHL-producing bacteria have been identified in over 37 genera within the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria (9,11,14,32) and the Cyanobacteria (10). In these bacteria, AHL-dependent QS systems have been shown to regulate many bacterial behaviors, such as virulence (8, 48) and biofilm formation (24, 29), mainly in response to cell densities. Therefore, AHL-dependent QS systems are now recognized for playing important roles in the regulation of bacterial behavior.The AHL-based QS systems usually contain a luxI gene homologue, responsible for the synthesis of AHLs, and a luxR gene homologue, an AHL-dependent transcriptional regulator (19). The LuxI/LuxR-type QS systems have been experimentally identified and studied in more than 70 different species in the phylum Proteobacteria (6, 15)...

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Section: Methodsmentioning

confidence: 99%

“…The metagenomic libraries were constructed from activated sludge of a coke plant wastewater treatment system containing various organic pollutants (22,44) and forest soil as a source of phylogenetically diverse microbes. An intercellular screening method was used, and GFP fluorescence of the biosensor was detected using a spectrofluorometer.…”

mentioning

confidence: 99%

Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach

Nasuno

Kimura

Fujita

et al. 2012

Appl Environ Microbiol

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“…In addition to having variations in symbiosis (24, 39, 60), various members of the Bradyrhizobiaceae , including Bradyrhizobium , harbor diverse biochemical functions such as photosynthesis, nitrification, sulfur oxidation, aromatic degradation, and oligotrophy (22, 25, 26, 33, 34, 45, 56, 68, 70). Thus, a long-standing question is how the members of the Bradyrhizobiaceae have acquired these diverse biochemical features.…”

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confidence: 99%

Complete Genome Sequence of <i>Bradyrhizobium</i> sp. S23321: Insights into Symbiosis Evolution in Soil Oligotrophs

et al. 2012

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Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.

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“…One of these methods, the function-based screening method, is generally selected based on the enzymatic activity of novel gene products in the metagenomic library. [34][35][36] In this method, environmental DNAs are cloned into expression vectors and propagated in appropriate hosts, followed by evaluating the activity expressed by the recombinants. [37][38][39][40] The evaluation of activity is often performed using a sensor strain or an indicator substrate that shows a color change as a result of biocatalytic conversion on agar plates.…”

Section: Metagenomics: Access To Uncultured Bacteriamentioning

confidence: 99%

“…metagenomic libraries were constructed from the activated sludge of a coke plant wastewater treatment system containing various organic pollutants 39,45 and forest soil as a source of phylogenetically diverse microbes. An "intercellular" screening method was used, and GFP fluorescence of the biosensor was detected using a spectrofluorometer (Fig.…”

Section: Screening For Luxi/luxr-type Qs Quorum-sensing System From Amentioning

confidence: 99%

Isolation and Characterization of a 4-Nitrotoluene-Oxidizing Enzyme from Activated Sludge by a Metagenomic Approach

Cited by 20 publications

References 49 publications

Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach

Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach

Complete Genome Sequence of <i>Bradyrhizobium</i> sp. S23321: Insights into Symbiosis Evolution in Soil Oligotrophs

Metagenomic approaches to understanding phylogenetic diversity in quorum sensing

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