Attenuation of Quorum Sensing Regulated Virulence of Pectobacterium carotovorum subsp. carotovorum through an AHL Lactonase Produced by Lysinibacillus sp. Gs50

Abstract

Quorum sensing (QS) is a mechanism in which Gram negative bacterial pathogens sense their population density through acyl homoserine lactones (AHLs) and regulate the expression of virulence factors. Enzymatic degradation of AHLs by lactonases, known as quorum quenching (QQ), is thus a potential strategy for attenuating QS regulated bacterial infections. We characterised the QQ activity of soil isolate Lysinibacillus sp. Gs50 and explored its potential for controlling bacterial soft rot of crop plants. Lysinibacillus sp. Gs50 inactivated AHL, which could be restored upon acidification, suggested that inactivation was due to the lactone ring hydrolysis of AHL. Heterologous expression of cloned gene for putative hydrolase (792 bp) designated adeH from Lysinibacillus sp. Gs50 produced a ~29 kDa protein which degraded AHLs of varying chain length. Mass spectrometry analysis of AdeH enzymatic reaction product revealed that AdeH hydrolyses the lactone ring of AHL and hence is an AHL lactonase. Multiple sequence alignment of the amino acid sequence of AdeH showed that it belongs to the metallo- β- lactamase superfamily, has a conserved "HXHXDH" motif typical of AHL lactonases. KM for AdeH for C6HSL was found to be 3.089 μM and the specific activity was 0.8 picomol min-1μg-1. AdeH has not so far been reported from any Lysinibacillus sp. and has less than 40% identity with known AHL lactonases. Finally we found that Lysinibacillus sp. Gs50 can degrade AHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc), a common cause of soft rot. This QQ activity causes a decrease in production of plant cell wall degrading enzymes of Pcc and attenuates symptoms of soft rot in experimental infection of potato, carrot and cucumber. Our results demonstrate the potential of Lysinibacillus sp. Gs50 as a preventive and curative biocontrol agent.

Fig 1. Characterization of AHL degrading Lysinibacillus sp. Gs50.

(a) AHL degrading enzyme in Lysinibacillus sp. Gs50 cells. 25μM C6HSL was added to culture supernatant (A 1, 2, 3), sonicated pellet (B 1, 2, 3), supernatant after sonication (C 1, 2, 3) and whole cell of Lysinibacillus sp. Gs50 (D 1, 2, 3). C. violaceum CV026 (E 1, 2, 3) + 25μM C6HSL is no treatment control and only C. violaceum CV026 (F 1, 2, 3) is biosensor control (b) Growth of Lysinibacillus sp. Gs50 in minimal medium containing C6HSL as sole carbon source assessed based on OD₆₀₀. Values are represented as mean ±SD for three replicates (c) Recovery of Lysinibacillus sp. Gs50 degraded C6HSL by acidification. Reaction mixture of Lysinibacillus sp. Gs50 + 25μM C6HSL (A 1, 2, 3) and acidified reaction mixture of Lysinibacillus sp. Gs50 + 25μM C6HSL with 25μl of 50mM HCl (B 1, 2, 3). C. violaceum CV026 + 25μM C6HSL (C 1, 2, 3) and acidified C. violaceum CV026 + 25μM C6HSL (D 1, 2, 3) are controls.

Fig 2. Characterization of E.coli BL21(DE3) pET22b(+)/adeH.

(a) SDS PAGE of E.coli BL21(DE3) pET22b(+) expressing AdeH. Lane 1: E.coli BL21(DE3) lysate, Lane 2: Lysate of E.coli BL21(DE3) pET22b(+) without IPTG induction, Lane 3: Lysate of E.coli BL21(DE3) pET22b(+) with IPTG induction, Lane 4: Lysate of E.coli BL21(DE3) pET22b(+)/adeH without IPTG induction, Lane 5: Lysate of E.coli BL21(DE3) pET22b(+)/adeH with IPTG induction, M: Protein molecular weight marker. (b) AHL degradation assay of E.coli BL21(DE3) pET22b(+)/adeH. 25μM C6HSL was added to Lysinibacillus sp. Gs50 (A 1, 2, 3), E.coli BL21(DE3) (B 1, 2, 3), E.coli BL21(DE3) pET22b(+) without IPTG induction (C 1, 2, 3), E.coli BL21(DE3) pET22b(+) with IPTG induction (D 1, 2, 3), E.coli BL21(DE3) pET22b(+)/adeH without IPTG induction (E 1, 2, 3), E.coli BL21 (DE3) pET22b(+)/adeH with IPTG induction (F 1, 2, 3). C. violaceum CV026 (G 1, 2, 3) + 25μM C6HSL is no treatment control and only C. violaceum CV026 (H 1, 2, 3) is biosensor control. (c) Soft rot attenuation assay of E.coli BL21 (DE3) pET22b(+)/adeH caused by PccBR1. Potato slices were inoculated with A: PccBR1, B: IPTG induced E.coli BL21(DE3) pET22b(+) and PccBR1, C: IPTG induced E.coli BL21 (DE3) pET22b(+)/adeH and PccBR1, D: PBS control.

Fig 3. Mechanism of AdeH catalysis.

(a) SDS PAGE analysis of purified His₆ tagged AdeH. Lane 1: Unbound Fraction, Lane 2: First wash fraction, Lane 3: Last wash fraction, Lane 4: First elution fraction (AdeH), Lane 5: Second elution fraction (AdeH), Lane 6: Third elution fraction (AdeH), M: Protein molecular weight marker (b) AHL degradation assay of purified AdeH. 30 μM C6HSL treated with 100 μg of purified and acetone precipitated AdeH (A 1, 2, 3, 4, 5) and no treatment control of 30 μM C6HSL (B 1, 2, 3, 4, 5) were exposed to C. violaceum CV026 (c) ESI-MS analysis of C6HSL (d) ESI-MS analysis of C6HSL degraded by AdeH showing a peak of C6HS.

Fig 4. Relationship of AdeH with other AHL lactonases.

(a) Multiple sequence alignment of AdeH and other representative AHL lactonases. Sequence alignment was performed by Clustal Omega online software. Representative AHL lactonases are AidC (Accession: BAM28988), MomL (Accession: AIY30473), AiiB (Accession: NP_396590), AhlD (Accession: AAP57766.1), AiiA (Accession: AAF62398.1), AttM (Accession: AAD43990.1) and AhlK (Accession: AAO47340.1). The shared “HXHXDH” motifs highlighted in bold. Asterisk indicates positions which have a single, fully conserved residue. Colon indicates conservation between groups of strongly similar properties. Period indicates conservation between groups of weakly similar properties. (b) Neighbor-joining tree of AHL lactonases belonging to the metallo- β-lactamase, phosphotriesterase and α/β hydrolase-fold family based on amino acid sequences. The dendrogram was constructed by neighbor joining method with the ClustalW program in the MEGA 7 software package (1,000 bootstrap replicates). Scale bar, 0.2 substitutions per amino acid position.

Fig 5. Biochemical characterization of AdeH.

(a) Effect of Temperature on AdeH activity (b) Effect of pH on AdeH activity (c) Thermal stability of AdeH (d) Effect of cations and EDTA on AdeH activity. Values represent the mean of three replications. Bars indicate standard deviation of the mean.

Fig 6. In vitro co-culture of Lysinibacillus sp. Gs50 and PccBR1.

(a) Influence of Lysinibacillus sp. Gs50 on the growth of PccBR1 in co-culture. (b) 3OC6HSL production by PccBR1 in the co-culture with Lysinibacillus sp. Gs50. Presence of 3OC6HSL is indicated by purple zone produced by biosensor C. violaceum CV026 around the well (c) Diameter of the purple zone in mm from the supernatant of Lysinibacillus sp. Gs50 and PccBR1 co-culture and PccBR1 alone (d) Polygalacturonase (PG) activity in the supernatant of Lysinibacillus sp. Gs50 and PccBR1 co-culture (e) Pectin lyase (PNL) activity in the supernatant of Lysinibacillus sp. Gs50 and PccBR1 co-culture. Values represent the mean of three replications. Bars indicate standard deviation of the mean. Statistical analysis was done using two-way unpaired t- test (** = p < 0.01).

Fig 7. Quorum quenching based biocontrol potential of Lysinibacillus sp. Gs50 against soft rot causing PccBR1.

(a) Soft rot attenuation assay on different hosts (Potato, Carrot, Cucumber) of PccBR1, co-inoculated with Lysinibacillus sp. Gs50 and PccBR1. PccBR1 alone is pathogen control and PBS is no infection control (b) Curative and preventive quorum quenching based biocontrol potential of Lysinibacillus sp. Gs50. In curative soft rot attenuation assay the pathogen PccBR1 was inoculated 12 hours before Lysinibacillus sp. Gs50 on potato slices. In preventive soft rot attenuation assay Lysinibacillus sp. Gs50 was inoculated 12 hours before the pathogen PccBR1. PBS inoculated slices were taken as no infection control.