A probable aculeacin A acylase from the Ralstonia solanacearum GMI1000 is N-acyl-homoserine lactone acylase with quorum-quenching activity.
BACKGROUND
The infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by N-acylhomoserine lactones (AHLs) acting as signal molecules. AHL-acylase is a quorum quenching enzyme and degrades AHLs by removing the fatty acid side chain from the homoserine lactone ring of AHLs. This blocks AHL accumulation and pathogenic phenotypes in quorum sensing bacteria.


RESULTS
An aac gene of undemonstrated function from Ralstonia solanacearum GMI1000 was cloned, expressed in Escherichia coli; it inactivated four AHLs that were tested. The sequence of the 795 amino acid polypeptide was considerably similar to the AHL-acylase from Ralstonia sp. XJ12B with 83% identity match and shared 39% identity with an aculeacin A acylase precursor from the gram-positive actinomycete Actinoplanes utahensis. Aculeacin A is a neutral lipopeptide antibiotic and an antifungal drug. An electrospray ionisation mass spectrometry (ESI-MS) analysis verified that Aac hydrolysed the amide bond of AHL, releasing homoserine lactone and the corresponding fatty acids. However, ESI-MS analysis demonstrated that the Aac could not catalyze the hydrolysis of the palmitoyl moiety of the aculeacin A. Moreover, the results of MIC test of aculeacin A suggest that Aac could not deacylate aculeacin A. The specificity of Aac for AHLs showed a greater preference for long acyl chains than for short acyl chains. Heterologous expression of the aac gene in Chromobacterium violaceum CV026 effectively inhibited violacein and chitinase activity, both of which were regulated by the quorum-sensing mechanism. These results indicated that Aac could control AHL-dependent pathogenicity.


CONCLUSIONS
This is the first study to find an AHL-acylase in a phytopathogen. Our data provide direct evidence that the functioning of the aac gene (NP520668) of R. solanacearum GMI1000 is via AHL-acylase and not via aculeacin A acylase. Since Aac is a therapeutic potential quorum-quenching agent, its further biotechnological applications in agriculture, clinical and bio-industrial fields should be evaluated in the near future.
Chen
Chin-Nung
CN
Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan, ROC. d91623801@ntu.edu.tw
Chen
Chii-Jaan
CJ
Liao
Chen-Ting
CT
Lee
Chia-Yin
CY
D054742:Acyl-Butyrolactones / Q000378:metabolism*; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002861:Chromobacterium / Q000378:metabolism; D003001:Cloning, Molecular; D008826:Microbial Sensitivity Tests; D010456:Peptides, Cyclic / Q000378:metabolism; D053038:Quorum Sensing; D043368:Ralstonia solanacearum / Q000201:enzymology* / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
D054742:Acyl-Butyrolactones; D001426:Bacterial Proteins; D010456:Peptides, Cyclic; C011634:aculeacin A; D000581:Amidohydrolases; C060671:aculeacin A acylase