Nobutaka Someya

AiiM, a Novel Class of N-Acylhomoserine Lactonase from the Leaf-Associated Bacterium Microbacterium testaceum

ABSTRACT N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many Gram-negative bacteria. We have reported that Microbacterium testaceum StLB037, which was isolated from the leaf surface of potato, has AHL-degrading activity. In this study, we cloned the aiiM gene from the genomic library of StLB037, which has AHL-degrading activity and shows high homology with the α/β hydrolase fold family from Actinobacteria. Purified AiiM as a maltose binding fusion protein showed high degrading activity of AHLs with both short- and long-chain AHLs with or without substitution at carbon 3. High-performance liquid chromatography analysis revealed that AiiM works as an AHL lactonase that catalyzes AHL ring opening by hydrolyzing lactones. In addition, expression of AiiM in the plant pathogen Pectobacterium carotovorum subsp. carotovorum reduced pectinase activity markedly and attenuated soft rot symptoms on potato slices. In conclusion, this study indicated that AiiM might be effective in quenching quorum sensing of P. carotovorum subsp. carotovorum.

Synergistic Antifungal Activity of Chitinolytic Enzymes and Prodigiosin Produced by Biocontrol Bacterium, Serratia marcescens Strain B2 against Gray Mold Pathogen, Botrytis cinerea

Serratia marcescensstrain B2 is an effective biocontrol agent against gray mold of cyclamen, caused by Botrytis cinerea Persoon. Strain B2 has strong antifungal activity against B. cinerea in vitro. The culture filtrate was found to contain different types of chitinolytic enzyme activity, including endochitinase and chitobiase activity. Four chitinolytic enzymes were detected among the extracellular proteins of strain B2. Two major enzymes, a 58-kDa endochitinase and a 98-kDa chitobiase, were purified by chromatography and electrofocusing, respectively. Both enzymes inhibited spore germination of B. cinerea. And a red pigment, prodigiosin, extracted and purified from the bacterial cells, also inhibited spore germination. When prodigiosin and the chitinolytic enzymes were applied in concert, a synergistic inhibitory effect was observed. S. marcescens strain B2 has multiple modes of action against the pathogen.

Genome Sequence of Microbacterium testaceum StLB037, an N-Acylhomoserine Lactone-Degrading Bacterium Isolated from Potato Leaves

Microbacterium testaceum is an endophytic Gram-positive bacterium that resides within plant hosts. M. testaceum StLB037 was isolated from potato leaves and shows N-acylhomoserine lactone-degrading activity. Here, we present the 3.98-Mb complete genome sequence of StLB037, with an average GC content of 70.28%.

Novel N-Acylhomoserine Lactone-Degrading Bacteria Isolated from the Leaf Surface of Solanum tuberosum and Their Quorum-Quenching Properties

We isolated and identified AHL-degrading bacteria from the leaf surface of Solanum tuberosum. The 16 isolates inactivated both short- and long-chain AHLs. Two of these isolates, identified as Microbacterium testaceum, showed putative AHL-lactonase activity. These two strains interrupted quorum-sensing dependent bacterial infection by plant pathogen Pectobacterium carotovorum subsp. carotovorum. M. testaceum strains, which were isolated in this study, might be useful in the biocontrol of plant diseases.

AidC, a novel N-acylhomoserine lactonase from the potato root-associated cytophaga-flavobacteria-bacteroides (CFB) group bacterium Chryseobacterium sp. strain StRB126.

ABSTRACT N-Acylhomoserine lactones (AHLs) are used as quorum-sensing (QS) signal molecules by many Gram-negative bacteria. We have reported that Chryseobacterium sp. strain StRB126, which was isolated from the root surface of potato, has AHL-degrading activity. In this study, we cloned and characterized the aidC gene from the genomic library of StRB126. AidC has AHL-degrading activity and shows homology to several metallo-β-lactamase proteins from Bacteroidetes, although not to any known AHL-degrading enzymes. Purified AidC, as a maltose-binding fusion protein, showed high degrading activity against all tested AHLs, whether short- or long-chain forms, with or without substitution at carbon 3. High-performance liquid chromatography (HPLC) analysis revealed that AidC functions as an AHL lactonase catalyzing AHL ring opening by hydrolyzing lactones. An assay to determine the effects of covalent and ionic bonding showed that Zn2+ is important to AidC activity both in vitro and in vivo. In addition, the aidC gene could also be PCR amplified from several other Chryseobacterium strains. In conclusion, this study indicated that the aidC gene, encoding a novel AHL lactonase, may be widespread throughout the genus Chryseobacterium. Our results extend the diversity and known bacterial hosts of AHL-degrading enzymes.

Complete genome sequence and characterization of the N-acylhomoserine lactone-degrading gene of the potato leaf-associated Solibacillus silvestris

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many gram-negative bacteria. We have reported that Solibacillus silvestris, which was isolated from the potato leaf, has AHL-degrading activity. To identify the AHL-degrading gene, whole genome sequencing of S. silvestris StLB046 was performed by using pyrosequencing technology. As the result of the BLAST search, one predicted ORF (ahlS) showed slight similarity to AiiA-like AHL lactonase from Bacillus cereus group. Escherichia coli harboring the ahlS-expressing plasmid showed high AHL-degrading activity. The ahlS-cording region was also amplified by PCR from the other potato leaf-associated and AHL-degrading S. silvestris strains. Purified AhlS as a maltose binding fusion protein showed high AHL-degrading activity and catalyzes AHL ring opening by hydrolyzing lactones. In addition, expression of ahlS in plant pathogen Pectobacterium carotovorum subsp. carotovorum attenuated maceration of the potato slices. Our results suggest that AHL-degrading activity of ahlS might perform useful functions such as useful biocontrol agents.

Potential of Serratia marcescens strain B2 for biological control of rice sheath blight

Serratia marcescens strain B2 inhibited mycelial growth of the rice sheath blight pathogen Rhizoctonia solani AG-1 IA. Rice plants were treated with bacterial suspension and then challenge inoculated with the pathogen. Application of S. marcescens effectively reduced the incidence of sheath blight. S. marcescens survived in soil under glasshouse conditions at ca. 108 colony forming units g−1 of soil for 4 weeks after application. These results suggest that S. marcescens has potential as an effective and persistent biological control agent for rice sheath blight.

Induced resistance to rice blast by antagonistic bacterium, Serratia marcescens strain B2

An antagonistic bacterium, Serratia marcescens strain B2, controlled rice blast after being sprayed onto rice phylloplane, as did the bacterial suspension when poured into rhizosphere soil of rice plants. Three days after root treatment, rice blast conidia were sprayed onto rice foliage. A week after pathogen inoculation, rice blast was suppressed and lesions caused by the pathogen decreased in size. Brown deposits were observed around sites of pathogen infection after root treatment. Induced resistance was not associated with an increase in the activitiy of peroxidase, phenylalanine ammonia lyase, tyrosine ammonia lyase, β-1,3-glucanase, β-1,4-glycosidase, N-acetylhexosaminidase or chitinase. However, lipoxygenase levels were elevated after the root treatment with strain B2 following inoculation with the pathogen. Strain B2 was not detected in rice foliage after root treatment. These data suggest that strain B2 induced resistance against rice blast caused by Pyricularia oryzae.

Phenazine antibiotic production and antifungal activity are regulated by multiple quorum-sensing systems in Pseudomonas chlororaphis subsp. aurantiaca StFRB508.

A number of gram-negative bacteria have a quorum-sensing system and produce the N-acylhomoserine lactone (AHL) as a signal molecule. Pseudomonas chlororaphis subsp. aurantiaca StFRB508 produces one of the phenazine derivatives, phenazine-1-carboxylic acid (PCA). Whole-genome sequencing of StFRB508 revealed the presence of two sets of AHL-synthase and AHL-receptor gene, phzIR and aurIR. The mutation of phzI drastically decreased PCA production, but the mutation of aurI did not affect PCA production. The phzI and aurI double mutant did not show any PCA production. StFRB508 produces three major AHLs, N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). As the results of TLC analysis, PhzI mainly catalyzes the biosynthesis of 3-hydroxy-C6-HSL, and AurI catalyzes the biosynthesis of C4-HSL and C6-HSL. PCA production in the phzI and aurI double mutant was restored by exogenous AHLs and the most active AHL was 3-hydroxy-C6-HSL. StFRB508 showed high inhibitory activity of the development of mycelia of plant pathogenic fungi, Fusarium oxysporum f. sp. conglutinans. However, the phzI and aurI double mutant could not inhibit the development of mycelia. These results demonstrated that the multiple quorum-sensing system play an important role in PCA production and antifungal activity in StFRB508.

Complete genome sequence of the biocontrol strain Pseudomonas protegens Cab57 discovered in Japan reveals strain-specific diversity of this species.

The biocontrol strain Pseudomonas sp. Cab57 was isolated from the rhizosphere of shepherd’s purse growing in a field in Hokkaido by screening the antibiotic producers. The whole genome sequence of this strain was obtained by paired-end and whole-genome shotgun sequencing, and the gaps between the contigs were closed using gap-spanning PCR products. The P. sp. Cab57 genome is organized into a single circular chromosome with 6,827,892 bp, 63.3% G+C content, and 6,186 predicted protein-coding sequences. Based on 16S rRNA gene analysis and whole genome analysis, strain Cab57 was identified as P. protegens. As reported in P. protegens CHA0 and Pf-5, four gene clusters (phl, prn, plt, and hcn) encoding the typical antibiotic metabolites and the reported genes associated with Gac/Rsm signal transduction pathway of these strains are fully conserved in the Cab57 genome. Actually strain Cab57 exhibited typical Gac/Rsm activities and antibiotic production, and these activities were enhanced by knocking out the retS gene (for a sensor kinase acting as an antagonist of GacS). Two large segments (79 and 115 kb) lacking in the Cab57 genome, as compared with the Pf-5 genome, accounted for the majority of the difference (247 kb) between these genomes. One of these segments was the complete rhizoxin analog biosynthesis gene cluster (ca. 79 kb) and another one was the 115-kb mobile genomic island. A whole genome comparison of those relative strains revealed that each strain has unique gene clusters involved in metabolism such as nitrite/nitrate assimilation, which was identified in the Cab57 genome. These findings suggest that P. protegens is a ubiquitous bacterium that controls its biocontrol traits while building up strain-specific genomic repertoires for the biosynthesis of secondary metabolites and niche adaptation.