A novel manganese efflux system, YebN, is required for virulence by Xanthomonas oryzae pv. oryzae

Abstract

Manganese ions (Mn(2+)) play a crucial role in virulence and protection against oxidative stress in bacterial pathogens. Such pathogens appear to have evolved complex mechanisms for regulating Mn(2+) uptake and efflux. Despite numerous studies on Mn(2+) uptake, however, only one efflux system has been identified to date. Here, we report on a novel Mn(2+) export system, YebN, in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight. Compared with wild-type PXO99, the yebN mutant was highly sensitive to Mn(2+) and accumulated high concentrations of intracellular manganese. In addition, we found that expression of yebN was positively regulated by Mn(2+) and the Mn(2+)-dependent transcription regulator, MntR. Interestingly, the yebN mutant was more tolerant to methyl viologen and H(2)O(2) in low Mn(2+) medium than PXO99, but more sensitive in high Mn(2+) medium, implying that YebN plays an important role in Mn(2+) homoeostasis and detoxification of reactive oxygen species (ROS). Notably, deletion of yebN rendered Xoo sensitive to hypo-osmotic shock, suggesting that YebN may protect against such stress. That mutation of yebN substantially reduced the Xoo growth rate and lesion formation in rice implies that YebN could be involved in Xoo fitness in host. Although YebN has two DUF204 domains, it lacks homology to any known metal transporter. Hence, this is the first report of a novel metal export system that plays essential roles in hypo-osmotic and oxidative stress, and virulence. Our results lay the foundations for elucidating the complex and fascinating relationship between metal homeostasis and host-pathogen interactions.

Figure 1. YebN is a conserved integral membrane protein with two DUF204 domains.

(A) Domain organization of the Xoo YebN. Two DUF204 domains exist in YebN (http://pfam.sanger.ac.uk/family?acc=PF02659). (B) Alignment of two DUF204 domains (13–81 and 119–182) of YebN and the DUF204 conserved sequence. (C) Protein secondary structure was predicted by using Split-4.0 (http://split.pmfst.hr/split/4/). Red line: transmembrane helix preference; Blue line: beta preference; Black line: modified hydrophobic moment index; Maroon boxes (below abscisa): predicted transmembrane (TM) helix position. YebN contains six transmembrane (TM) helixes (TM1, TM2, TM3, TM4, TM5 and TM6). (D) A Western blot of Xoo cells (C-ΔyebN-His) total membrane proteins (TM), cytoplasmic fraction (CP), inner membrane fraction (IM) and outer membrane fraction (OM) was probed for subcellular location of YebN, using His tag antibodies. An equal amount (2 µg) of protein was loaded in each lane. The blot is representative of three independent experiments. (E) Sequence alignment of Xoo YebN against other bacterial homologues. X. oryzae: Xanthomonas oryzae pv. oryzae str. PXO99; X. campestris: Xanthomonas campestris pv. campestris str. 8004; P. syringae: Pseudomonas syringae pv. tomato str. DC3000; E. coli: Escherichia coli str. K-12 substr. MG1655; B. subtilis: Bacillus subtilis subsp. subtilis str. 168; Y. enterocolitica: Yersinia enterocolitica subsp. enterocolitica 8081; S. enterica: Salmonella enterica subsp. enterica serovar Heidelberg str. SL486. Alignments (B, E) were performed using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The homology between the proteins (B, E) is indicated as follows: *, fully conserved residues; :, closed conservative substitutions; conservative substitutions.

Figure 2. YebN is involved in manganese efflux in Xoo.

(A) Phenotypes of wild type cells (top row), ΔyebN cells (middle row) and the complemented strain C-ΔyebN cells (bottom row) on PGA plates. Each spot was inoculated from 2 µl of a 10-fold dilution series from stationary cells (i.e., 10⁰, 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴ fold from left to right). (B) The phenotypes observed from growth in liquid minimal medium and the effects of exogenous manganese in wild type and ΔyebN cells. (C) The cellular manganese content of the wild type and the yebN mutant grown in low- or high-manganese concentration. Cells were grown in media supplemented with 0 mM (L) or 0.15 mM MnSO₄ (H). The manganese content of whole cells was determined by absorption spectroscopy. The data shown represents the mean ± standard deviations (SD) from four independent experiments (*P<0.01).

Figure 3. Amino acid substitutes in YebN cytoplasmic regions alter Xoo Mn²⁺ tolerance.

The strains that yebN mutant (ΔyebN) carries extrachromosomal yebN wild type sequence (WT) or some point mutations in YebN cytoplasmic regions (G25A, A26N, A27N, A27ND35A and G167A) cloned into the pHM1 vector were analyzed as described in Figure 2A. The strain that ΔyebN contains the pHM1 vector was the negative control (CK).

Figure 4. YebN functions in Escherichia coli are similar to those in Xoo.

The sensitivity of E. coli yebN mutant (JW5830) to exogenous Mn²⁺ and the complementary analysis of JW5830 by the Xoo homologue. NE9062 (pHM1) and JW5830 (pHM1) are wild type E. coli. MG1655 and the yebN mutant harbor pHM1 plasmids, respectively. JW5830 (pHM-yebN) is a yebN mutant containing the Xoo yebN gene in a pHM1 vector. The experimental protocol was the same as described in Figure 2A, except bacteria were cultured in LB medium at 37°C.

Figure 5. Mn²⁺ up-regulates the expression of yebN via MntR.

(A) Wild type strains containing the yebN promoter gusA fusion were grown in M4 medium with or without the indicated ions. Cells were harvested by centrifugation and GUS activity was measured as described elsewhere . (B) yebN expression (GUS activity) in wild type strains grown in PGA medium cantaining different concentration of Mn²⁺, Ca²⁺ or Fe²⁺. (C) The yebN expression in the mntR mutant in PSA medium cantaining different concentration of Mn²⁺ (*P<0.01). (D) The effect of the MntR binding site mutation in the yebN promoter on yebN expression. Error bars correspond to standard deviations (*P<0.01). (E) EMSA showing in vitro binding of MntR to the yebN promoter. His-tag Xoo MntR protein purified from E. coli BL21 (DE3) and the 95 bp ³²P-labeled DNA fragment containing the predicted MntR binding site of the yebN promoter were used in the protein-binding assay. The ³²P-labeled 95 bp DNA fragment containing the putative MntR binding site mutation was used as a mutant probe and the unlabeled fragment used as a mutant competitor.

Figure 6. Mutation of yebN alters Xoo viability under oxidative stress.

(A) Strains were grown to an OD of 0.1 then treated with either hydrogen peroxide (10 mM) or methyl viologen (60 mM). Bacterial cfu were calculated immediately before adding oxidants and at 15 min (hydrogen peroxide) or 60 min (methyl viologen, MV) post-treatment using serial dilution estimates and direct counts. (B) Bacteria were treated with manganese (0, 0.1 or 0.5 mM) and methyl viologen (0 or 60 mM), and cfu calculated as described in panel A. Data represent the mean ± SD of the relative survival (% of 0 mM Mn²⁺) from three independent replicates (*P<0.05). (C) Hydrogen peroxide production by the wild type and yebN mutant. Bacteria were cultured in PSA media to an OD of 0.5 and then diluted 1∶20 into fresh PSA or PSA supplemented with 0.15 mM MnSO₄. After growth for 4 additional hours, a 2 ml culture was centrifuged and the pellet re-suspended in sterile PBS. Bacteria were incubated at room temperature for 30 min to allow H₂O₂ production. The data shown represents the mean ± SD of the relative H₂O₂ content (% of wild type) from three independent replicates (*P<0.05).

Figure 7. YebN protects bacteria against hypo-osmotic shock.

(A) Bacteria were cultured in PSA media to an OD600 of 1.0. A 1 ml culture was centrifuged, the pellet washed twice with PSA, and re-suspended in PSA. 20 µl of a 10-fold serial dilution (left: in PSA, right: in ddH₂O) of the cells was plated onto PSA plates. (B) Bacteria were cultured in PSA media to an OD600 of 0.5. A 1 ml culture was centrifuged, the pellet re-suspended in PSA and cfu calculated. An additional 1 ml culture was collected as above, washed twice with ddH₂O and re-suspended in 10 ml ddH₂O. Bacteria were incubated at room temperature for 60 min and enumerated by serial dilution. The data shown represents the mean ± SD from three independent replicates (*P<0.05).

Figure 8. YebN is involved in Xoo pathogenesis.

(A) Lesions on rice leaves inoculated with Xoo strains. Lane1: PXO99, lane2: ΔyebN, lane3: C-ΔyebN. 60-day-old susceptible rice cultivars (IR24) were tested. (B) Measurements of the lesion lengths obtained from 20 leaves at 2 weeks post-inoculation. Virulence assays were performed in triplicate and the Mean ± SD were calculated (*P<0.01). (C) Growth of Xoo strains in rice leaves. The mean CFU was calculated from three independent experiments using six leaves for each strain.

Figure 9. Schematic representation of the roles of YebN in virulence.

YebN is vital for manganese accumulation in bacterial cells (A) and possibly regulates cell membrane stability (F) that influences the bacterial hypo-osmotic response (G). Changes in intracellular manganese levels also regulate yebN expression (A) and alter its ability to protect bacteria against oxidative stress (B). Manganese and ROS might regulate its capacity to protect bacteria against hypotonic shock (H and I). Hypotonic shock might, in turn, influence ROS production and scavenge (I). Manganese, ROS and hypo-osmosis are all important factors that affect bacterial growth in the host (C, D and E). Solid lines indicate how the results of this study support the model; dashed lines indicate where no direct evidence has been obtained.