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 ␣/␤ 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.

b 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-␤-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 Zn 2؉ 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.

No abstract

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%.The genus Microbacterium belongs to the class of high-GCcontent Actinobacteria. Microbacterium testaceum is an endophytic bacterium that resides within plant hosts without causing disease symptoms (12). In many Gram-negative bacterial species, N-acylhomoserine lactone (AHL) has been identified as a signal compound involved in the quorum-sensing system (2). Many Gram-negative plant pathogens produce AHLs and regulate their virulence by AHL-mediated quorum sensing (10). We have reported the isolation of AHL-degrading M. testaceum strains from the leaf surface of the potato (6). We also cloned the aiiM gene from the genomic library of StLB037, which has AHL-lactonase activity and shows high homology with the ␣/␤ hydrolase fold family from Actinobacteria (11). To our knowledge, the complete genome sequence of the genus Microbacterium has not been deposited in the DDBJ/EMBL/GenBank databases at this point in time. In this work, we determined the genome sequence of M. testaceum StLB037.Single-and paired-end whole-genome shotgun sequencing of StLB037 was performed using Roche genome sequencer FLX Titanium pyrosequencing technology (5) provided by Operon Biotechnologies (Tokyo, Japan). We produced 584,678 reads, with an average read length of 287 bases. The total number of sequenced bases is 167,947,725, representing a sequencing depth of 42ϫ. With the use of Celera Assembler version 5.3, these reads were assembled into one large scaffold including 29 large contigs (Ͼ1,000 bp). Gap closure was attempted using gap-spanning clones and PCR products. Prediction of putative coding sequences and gene annotation were done using the Microbial Genome Annotation Pipeline (http://migap.lifesciencedb.jp/). Briefly, protein-coding sequences (CDSs) were predicted by the combined use of MetaGeneAnnotator (7), RNAmmer (3), tRNAScan (4), and BLAST (1).The complete genomic information for M. testaceum StLB037 is contained on a single circular chromosome of 3,982,034 bp with an average GC content of 70.28%. The genome contains 3,676 protein-coding genes, two rRNA operons, and 45 tRNA genes.The aiiM gene, which has been identified from the genomic library of StLB037, was found as a single copy in the complete genome. Many AHL-degrading genes have been cloned and characterized from various bacterial species (8). We searched for the homologs of the reported AHL-degrading genes in the complete genome of StLB037. As a result, one predicted CDS (MTES_1124), which encoded 309 amino acids, showed 26.3% identity to the reported AHL-degrading enzyme, QsdA (9). QsdA is a phosphotriesterase-like AHL lactonase from Rhodococcus erythropolis W2, which belongs to the class of Actinobacteria as well as M. testaceum.In summary, the AHL-degrading activi...

N-Acylhomoserine lactone (AHL)-degrading enzyme, AiiM, was identified from the potato leaf-associated Microbacterium testaceum StLB037. In this study, we cloned eight aiiM gene homologues from other AHL-degrading Microbacterium strains. The similarity of the chromosomal locus of the aiiM gene is associated with the phylogenetic classification based on 16S rRNA. Degenerate PCR revealed that the aiiM gene was only conserved in AHL-degrading Microbacterium strains, but not in fifteen Microbacterium type strains or two Microbacterium isolates from other plants. These results suggested that the high level of AHL-degrading activity in Microbacterium strains was caused by the aiiM gene encoded on their chromosome.