The Ti plasmid of Agrobacterium tumefaciens harbors an attM-paralogous gene, aiiB, also encoding N-Acyl homoserine lactonase activity

Abstract

The Agrobacterium tumefaciens C58 genome contains three putative N-acyl homoserine lactone (acyl-HSL) hydrolases, which are closely related to the lactonase AiiA of Bacillus. When expressed in Escherichia coli, two of the putative acyl-HSL hydrolases, AttM and AiiB, conferred the ability to degrade acyl-HSLs on the host. In Erwinia strain 6276, the lactonases reduced the endogenous acyl-HSL level and the bacterial virulence in planta.

FIG. 1.

Phylogenetic analysis of the putative AiiA homologues. In addition to the three A. tumefaciens C58 sequences, the putative AiiA homologues were from Bacillus halodurans C-125 (Ba.ha.gi15615822), Bradyrhizobium japonicum USDA110 (Br.ja.gi6655034), Deinococcus radiodurans R1 (De.ra.gi15805209 and De.ra.gi15806823), Listeria innocua Clip11262 (Li.in.gi16800723), Mesorhizobium loti MAFF303099 (Me.lo.gi13475678), Plesiomonas sp. strain M6 (Ps.sp.gi13173397), Sinorhizobium meliloti 1021 (Si.me.gi15964512), Staphylococcus aureus Mu50 (St.au.gi15924737), and Xylella fastidiosa 9a5c (Xy.fa.gi15837962). The glyoxalase GloB (Ec.co.gi2494853) of E. coli was used as an outgroup sequence. The sequences were aligned with the ClustalW program, and the phylogenetic tree was constructed by the neighbor-joining method. Only bootstrap values greater than 750 are shown. The taxonomic position refers to the α and γ subdivisions of the Proteobacteria (α-Proteo. and γ-Proteo., respectively), the Firmicutes (Firm.), and the Deinococcus-Thermus group (Deino./Therm.).

FIG. 2.

Conserved regions in the amino acid sequences of the putative AiiA homologues. The sequences of putative AiiA homologues from Deinococcus radiodurans R1 (De.ra.gi15805209 and De.ra.gi15806823), Xylella fastidiosa 9a5c (Xy.fa.gi15837962), Mesorhizobium loti MAFF303099 (Me.lo.gi13475678), Staphylococcus aureus Mu50 (St.au.gi15924737), Listeria innocua Clip11262 (Li.in.gi16800723), Bacillus halodurans C-125 (Ba.ha.gi15615822), Sinorhizobium meliloti 1021 (Si.me.gi15964512), and Plesiomonas sp. strain M6 (Ps.sp.gi13173397) and the sequences of glyoxalase GloB (Ec.co.gi2494853) of E. coli and AiiA from Bacillus sp. strain 240B1, AiiA_soil, AttM, AiiB, and AiiC from A. tumefaciens (Ag.tu.) are shown. Each one of the five segments that are conserved among the Zn metallohydrolases (2) was present in the AiiA homologues. When more than 10 sequences (of 14) contain an identical (or a physiochemically similar residue) at a given site, the consensus residue (or the abbreviation of their physiochemical family) is reported below the alignment as follows: h for the hydrophobic residues L, I, M, and V; s for the small hydrophobic residues S, P, T, A and G; and n for the negatively charged residues and their relatives, D, E, N, and Q. The residues that are conserved among the AiiA homologues and the GloB Zn metallohydrolase are shown in bold type in the GloB sequence, and the residues interacting with the metal cations are noted by an asterisk. Numbers in parentheses indicate the number of residues omitted in the sequences.

FIG. 3.

Kinetics of acyl-HSL degradation by the Agrobacterium AiiA family members. In cultures of E. coli harboring pMIR102 (attM) (▵), pMIR103 (aiiB) (⋄) and pMIR104 (aiiC) (□), the acyl-HSL concentration is expressed as a percentage of the initial concentration (25 μM) of C6-HSL and oxo-C8-HSL. The acyl-HSL degradation kinetics of control cultures of E. coli harboring p6010 and uninoculated medium supplemented with acyl-HSL were identical to those of E. coli harboring pMIR104. The experiments were done in triplicate, and the standard deviations (not shown) were always below 5%.

FIG. 4.

Oxo-C8-HSL levels in A. tumefaciens C58 and A. tumefaciens C58 strain lacking pAt. The black and grey bars show the oxo-C8-HSL levels in the culture supernatants of the wild-type A. tumefaciens C58 strain (□) and A. tumefaciens C58 strain lacking pAt (▵), respectively. The oxo-C8-HSL concentration in wild-type strain C58 was statistically lower (P < 0.05) than in the C58 strain lacking pAt. The experiments were done in duplicate, and the standard deviations (not shown) did not exceed 10% of the mean values. OD₆₀₀, optical density at 600 nm.

FIG. 5.

Acyl-HSL levels in E. carotovora subsp. atroseptica 6276 and lactonase-expressing derivatives. The ethyl acetate extracts obtained from E. carotovora subsp. atroseptica 6276 and its lactonase-expressing derivatives were serially diluted and spotted on TLC plates and then covered by Agrobacterium NTLR4 biosensor. In the presence of the AiiA, AttM, and AiiB lactonases, the levels of acyl-HSLs in the culture supernatant decreased 100- to 1,000-fold compared to the levels of E. carotovora subsp. atroseptica 6276 and E. carotovora subsp. atroseptica 6276 harboring p6010.

FIG. 6.

Maceration assays on potatoes. In each of the tubers inoculated with E. carotovora subsp. atroseptica 6276 harboring plasmid p6010 (19 tubers), pMIR101 (26 tubers), pMIR102 (24 tubers), or pMIR103 (25 tubers), the weight of macerated tissues was measured and classified in three categories: 0 to 50 mg (white bars), 51 to 400 mg (grey bars), and more than 401 mg (black bars). The data were collected from two independent experiments. Statistically different distributions of the maceration categories (α = 0.05, χ² test) are indicated by a different letter over the bar.