Satoshi Hanada

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

ABSTRACT 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 (C12-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.

Genome Sequence of the Multiple-β-Lactam-Antibiotic-Resistant Bacterium Acidovorax sp. Strain MR-S7.

ABSTRACT Acidovorax sp. strain MR-S7 was isolated from activated sludge in a treatment system for wastewater containing β-lactam antibiotic pollutants. Strain MR-S7 demonstrates multidrug resistance for various types of β-lactam antibiotics at high levels of MIC. The draft genome sequence clarified that strain MR-S7 harbors unique β-lactamase genes.

The effects of N-acylhomoserine lactones, β-lactam antibiotics and adenosine on biofilm formation in the multi-β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7

Bacteria in the natural ecosystem frequently live as adherent communities called biofilms. Some chemical compounds are known to affect biofilm formation. We investigated the effect of exogenous small molecules, N-acylhomoserine lactones (AHLs), β-lactam antibiotics, and adenosine, on biofilm formation in the β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7. Biofilm formation was induced by the addition of various types of AHL isomers and β-lactam antibiotics, whereas the addition of adenosine strongly interfered with the biofilm formation. A gene (macP) encoding adenosine deaminase (that converts adenosine to inosine controlling intracellular adenosine concentration) was successfully cloned from MR-S7 genome and heterologously expressed in Escherichia coli. The purified MacP protein clearly catalyzed the deamination of adenosine to produce inosine. A transcriptional analysis revealed that biofilm-inducing molecules, an AHL and a β-lactam antibiotic, strongly induced not only biofilm formation but also adenosine deaminase gene expression, suggesting that an elaborate gene regulation network for biofilm formation is present in the β-lactam antibiotic-resistant bacterium studied here.

Granulicella cerasi sp. nov., an acidophilic bacterium isolated from cherry bark.

A novel acidobacterial strain, Sakura1(T), was isolated from pieces of cherry bark. Cells of strain Sakura1(T) were non-motile, rod-shaped and stained Gram-negative. This strictly aerobic isolate was mesophilic but was able to grow at temperatures as low as 10 °C. Colonies were pink due to production of carotenoids, and its pigmentation was more pronounced in cells grown at lower temperature. This strain had endoglucanase activity. The main respiratory quinone was menaquinone-8, and major cellular fatty acids were iso-C(15 : 0), C(16 : 1)ω7c and C(16 : 0). Phylogenetic analysis of 16S rRNA gene sequences revealed that the strain was closely related to species of the genus Granulicella in subdivision 1 of the phylum Acidobacteria. The closest relative was Granulicella paludicola OB1010(T) (97.1% 16S rRNA gene sequence similarity). While common characteristics were found among the isolate and species of the genus Granulicella, there were obvious differences between them such as their cell morphology, cellulolytic activity, and tolerance to low temperature and NaCl concentration. Based on phylogenetic and phenotypic findings, a novel species is proposed in the genus Granulicella with the name Granulicella cerasi sp. nov. The type strain is Sakura1(T) ( = NBRC 107139(T) = DSM 23641(T)).

Metagenomic and biochemical characterizations of sulfur oxidation metabolism in uncultured large sausage-shaped bacterium in hot spring microbial mats.

So-called “sulfur-turf” microbial mats in sulfide containing hot springs (55–70°C, pH 7.3–8.3) in Japan were dominated by a large sausage-shaped bacterium (LSSB) that is closely related to the genus Sulfurihydrogenibium. Several previous reports proposed that the LSSB would be involved in sulfide oxidation in hot spring. However, the LSSB has not been isolated yet, thus there has been no clear evidence showing whether it possesses any genes and enzymes responsible for sulfide oxidation. To verify this, we investigated sulfide oxidation potential in the LSSB using a metagenomic approach and subsequent biochemical analysis. Genome fragments of the LSSB (a total of 3.7 Mb sequence including overlapping fragments) were obtained from the metagenomic fosmid library constructed from genomic DNA of the sulfur-turf mats. The sequence annotation clearly revealed that the LSSB possesses sulfur oxidation-related genes coding sulfide dehydrogenase (SD), sulfide-quinone reductase and sulfite dehydrogenase. The gene encoding SD, the key enzyme for sulfide oxidation, was successfully cloned and heterologously expressed in Escherichia coli. The purified recombinant enzyme clearly showed SD activity with optimum temperature and pH of 60°C and 8.0, respectively, which were consistent with the environmental conditions in the hot spring where the sulfur-turf thrives. Furthermore, the affinity of SD to sulfide was relatively high, which also reflected the environment where the sulfide could be continuously supplied. This is the first report showing that the LSSB harbors sulfide oxidizing metabolism adapted to the hot spring environment and can be involved in sulfide oxidation in the sulfur-turf microbial mats.

A Novel Quorum-Quenching N-Acylhomoserine Lactone Acylase from Acidovorax sp. Strain MR-S7 Mediates Antibiotic Resistance

ABSTRACT N-Acylhomoserine lactone acylase (AHL acylase) is a well-known enzyme responsible for disrupting cell-cell communication (quorum sensing) in bacteria. Here, we isolated and characterized a novel and unique AHL acylase (designated MacQ) from a multidrug-resistant bacterium, Acidovorax sp. strain MR-S7. The purified MacQ protein heterologously expressed in Escherichia coli degraded a wide variety of AHLs, ranging from C6 to C14 side chains with or without 3-oxo substitutions. We also observed that AHL-mediated virulence factor production in a plant pathogen, Pectobacterium carotovorum, was dramatically attenuated by coculture with MacQ-overexpressing Escherichia coli, whereas E. coli with an empty vector was unable to quench the pathogenicity, which strongly indicates that MacQ can act in vivo as a quorum-quenching enzyme and interfere with the quorum-sensing system in the pathogen. In addition, this enzyme was found to be capable of degrading a wide spectrum of β-lactams (penicillin G, ampicillin, amoxicillin, carbenicillin, cephalexin, and cefadroxil) by deacylation, clearly indicating that MacQ is a bifunctional enzyme that confers both quorum quenching and antibiotic resistance on strain MR-S7. MacQ has relatively low amino acid sequence identity to any of the known acylases (<39%) and has among the broadest substrate range. Our findings provide the possibility that AHL acylase genes can be an alternative source of antibiotic resistance genes posing a threat to human health if they migrate and transfer to pathogenic bacteria. IMPORTANCE N-Acylhomoserine lactones (AHLs) are well-known signal molecules for bacterial cell-cell communication (quorum sensing), and AHL acylase, which is able to degrade AHLs, has been recognized as a major target for quorum-sensing interference (quorum quenching) in pathogens. In this work, we succeeded in isolating a novel AHL acylase (MacQ) from a multidrug-resistant bacterium and demonstrated that the MacQ enzyme could confer multidrug resistance as well as quorum quenching on the host organism. Indeed, the purified MacQ protein was found to be bifunctional and capable of degrading not only various AHL derivatives but also multiple β-lactam antibiotics by deacylation activities. Although quorum quenching and antibiotic resistance have been recognized to be distinct biological functions, our findings clearly link the two functions by discovering the novel bifunctional enzyme and further providing the possibility that a hitherto-overlooked antibiotic resistance mechanism mediated by the quorum-quenching enzyme may exist in natural environments and perhaps in clinical settings.

In Situ Gene Expression Responsible for Sulfide Oxidation and CO2 Fixation of an Uncultured Large Sausage-Shaped Aquificae Bacterium in a Sulfidic Hot Spring.

We investigated the in situ gene expression profile of sulfur-turf microbial mats dominated by an uncultured large sausage-shaped Aquificae bacterium, a key metabolic player in sulfur-turfs in sulfidic hot springs. A reverse transcription-PCR analysis revealed that the genes responsible for sulfide, sulfite, and thiosulfate oxidation and carbon fixation via the reductive TCA cycle were continuously expressed in sulfur-turf mats taken at different sampling points, seasons, and years. These results suggest that the uncultured large sausage-shaped bacterium has the ability to grow chemolithoautotrophically and plays key roles as a primary producer in the sulfidic hot spring ecosystem in situ.