AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora

Abstract

N-acylhomoserine lactones, known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many gram-negative bacteria. AIs are involved in the regulation of diverse biological functions, including expression of pathogenic genes in the plant pathogens Pseudomonas solanacearum, several Erwinia species, and the human pathogen Pseudomonas aeruginosa. A bacterial isolate, Bacillus sp. 240B1, is capable of enzymatic inactivation of AIs. The gene (aiiA) for AI inactivation from Bacillus sp. 240B1 has been cloned and shown to encode a protein of 250 amino acids. Sequence alignment indicates that AiiA contains a "HXHXDH" zinc-binding motif that is conserved in several groups of metallohydrolases. Site-directed mutagenesis showed that conserved aspartate and most histidine residues are required for AiiA activity. Expression of aiiA in transformed Erwinia carotovora strain SCG1 significantly reduces the release of AI, decreases extracellular pectolytic enzyme activities, and attenuates pathogenicity on potato, eggplant, Chinese cabbage, carrot, celery, cauliflower, and tobacco. Our results indicate that the AI-inactivation approach represents a promising strategy for prevention of diseases in which virulence is regulated by AIs.

Figure 1

Deletion analysis of AI inactivation region of Bacillus sp. 240B1 (A) and the predicted amino acid sequence of the aiiA gene product (B). Clone E7-R3 is contained in cosmid vector pLAFR3, whereas the others are in cloning vector pGEM-7Zf(+). The location and direction of Plac promoters in the cosmid and in the pGEM-7Zf(+) clone are indicated by solid arrows. AI inactivation activity of the clones is shown in the second column: +, with AI inactivation activity; −, without AI inactivation activity. Restriction enzymes: E, EcoRI; H, HindIII; Ev, EcoRV; St, StyI. The open arrow indicates the location and transcription direction of the aiiA ORF. In the AiiA protein sequence, two conserved regions are boxed.

Figure 2

Time course of AI inactivation by purified AiiA protein. The purified AiiA protein was diluted to a concentration of 50 ng/μl with 1/15 M phosphate buffer (pH 8.0). Equal volume of diluted AiiA protein and 40 μM OHHL (⧫), ODHL (■), or OOHL (▴) was added in a 1.5-ml Eppendorf centrifuge tube. The reaction was conducted at 28°C. The same concentration of the denatured AiiA (boiling for 5 min) and OHHL in the phosphate buffer was used as control (●). Samples were taken at 10-min intervals for a total of 60 min, and the reaction was stopped by boiling for 3 min. The samples were assayed for AI activity as described (3).

Figure 3

Multiple alignment of the conserved regions among AiiA and other protein sequences. The numbering is based on the sequence of AiiA. The dashes indicate amino acids identical to AiiA sequence. The consensus amino acids are indicated. In species column, abbreviations are as follows: E, Escherichia; R, Rhodobacter; S, Schistosoma; B, Buchnera; Pse. carr., Pseudoalteromonas carrageenovora; M, Mycobacterium; C, Caenorhabditis; P, Pseudomonas; and Se, Serratia. In the protein column, the abbreviations are: Gly, glyoxalase II; Ary, arylsulfatase; Lac, β-lactamase.

Figure 4

Effect of aiiA gene product on cell growth (A), AI production (B), and extracellular pectolytic enzyme activity (C) in E. carotovora strains SCG1(E7-R3) (♦ in A and B, A in C), SCG1(pLAFR3) (■, R), and SCG1 (▴, S). The enzyme activities were expressed as the increase of absorbance at 235 nm (for Pel and Pnl) or 500 nm (for Peh) per minute per milliliter of culture supernatant.

Figure 5

Effect of aiiA gene expression on E. carotovora pathogenicity. From left to right, Chinese cabbage inoculated, respectively, with 10 μl of bacterial inoculum (2 × 10⁹ colony-forming units/ml) of SCG1(E7-R3), SCG1(pLAFR3), and SCG1. The photographs were taken 3 (Upper) and 6 (Lower) days after inoculation.