Norihiro Kato

In tests, Chromobacterium violaceum ATCC 12472 produced several N-acyl-L-homoserine lactones (AHLs). Of these, N-(3-hydroxydecanoyl)-L-homoserine lactone was dominant, and controlled violacein production by quorum sensing. Strain VIR07, an AHL-deficient mutant, did not produce violacein. Violacein production in VIR07 was induced by adding long-chain AHLs (C10-C16), but was inhibited by adding short-chain AHLs (C4-C8). Strain VIR07 showed the response of violacein production when AHLs diffused from a variety of AHL-producing bacteria on agar plates, and thus might be a useful biosensor for recognizing exogenous AHLs.

In this study, we have compared genomic DNA fingerprintings among isolates of methicillin-resistant Staphylococcus aureus (MRSA) by using pulsed-field gel electrophoresis (PFGE). Chromosomal fragments digested with SmaI were most suitable for the PFGE separation. SmaI cut genomic DNA into 15 to 20 fragments whose sizes ranged from about 30 to 1,500 kb. Thirty-one distinctive fragment patterns were identified in 111 infecting and colonizing MRSA isolates from six different hospitals in Japan. On the basis of the genomic typing by PFGE, we performed an epidemiological investigation of an outbreak of nosocomial MRSA infections among inpatients in Nagoya University Hospital. Ten types of chromosomal digestion were identified in the 20 strains isolated from 18 infected patients and 1 from colonized hospital personnel. According to the restriction patterns, we found that four types of these strains had caused epidemic infections among 13 patients in the outbreak. Two types (types 1 and 4) of the strains were involved in the death of five patients. The other infections were sporadic. The clarity and polymorphism of the chromosomal digestion patterns enabled us to discriminate between isolates which could not be differentiated by antibiogram or plasmid analysis. Classification of the genomic DNA fingerprinting patterns by PFGE is therefore proposed as a useful method for investigating the source, transmission, and spread of nosocomial MRSA infections.

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C(6)-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C(9)-CPA), had a strong inhibitory effect on prodigiosin production. C(9)-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C(9)-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C(6)-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C(9)-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many gram-negative bacteria. We have reported that Shewanella sp. strain MIB015 degrades AHLs. In the present study, we cloned the aac gene from MIB015 by PCR with specific primers based on the aac gene in Shewanella oneidensis strain MR-1, which showed high homology with the known AHL-acylases. Escherichia coli expressing Aac showed high degrading activity of AHLs with long acyl chains. HPLC analysis revealed that Aac worked as AHL-acylase, which hydrolyzed the amide bond of AHL. In addition, expression of Aac in fish pathogen Vibrio anguillarum markedly reduced AHL production and biofilm formation. In conclusion, this study indicates that Aac might be effective in quenching quorum sensing of fish pathogens.

A number of gram-negative bacteria have a quorum-sensing system and produce N-acyl-l-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. Pantoea ananatis is reported as a common colonist of wheat heads at ripening and causes center rot of onion. In this study, we demonstrated that P. ananatis SK-1 produced two AHLs, N-hexanoyl-l-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). We cloned the AHL-synthase gene (eanI) and AHL-receptor gene (eanR) and revealed that the deduced amino acid sequence of EanI/EanR showed high identity to those of EsaI/EsaR from P. stewartii. EanR repressed the ean box sequence and the addition of AHLs resulted in derepression of ean box. Inactivation of the chromosomal eanI gene in SK-1 caused disruption of exopolysaccharide (EPS) biosynthesis, biofilm formation, and infection of onion leaves, which were recovered by adding exogenous 3-oxo-C6-HSL. These results demonstrated that the quorum-sensing system involved the biosynthesis of EPS, biofilm formation, and infection of onion leaves in P. ananatis SK-1.