Hfq influences multiple transport systems and virulence in the plant pathogen Agrobacterium tumefaciens

Abstract

The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens.

Fig 1

Complex formation between Hfq, the sRNA AbcR1, and its target atu2422. (A) Gel retardation experiments with purified Hfq protein and AbcR1. ³²P-labeled AbcR1 sRNA (0.07 pmol) was incubated with increasing concentrations of Hfq protein at 30°C for 20 min. Final concentrations (fold excess) of added Hfq are shown above the gel. Lane 1 contains a water control. (B) Gel retardation experiments with AbcR1, Hfq, and the target atu2422 mRNA fragment (100 nucleotides). ³²P-labeled AbcR1 sRNA (0.07 pmol) was incubated with purified Hfq (1,000-fold excess) and increasing concentrations of atu2422. Final concentrations (fold excess) of added atu2422 RNA are shown above the gel. Lane 1 contains a water control. Samples were run on a 6% native gel at 250 V. The asterisks indicate radioactive (³²P) labeling of the sRNA AbcR1.

Fig 2

Effect of Hfq on AbcR1-mediated gene regulation in vivo. (A) Northern blot analysis of AbcR1 and atu2422 in A. tumefaciens wild type (WT) and the Δhfq strain. Hybridizations were performed with 8 μg of total RNA from cells grown in YEB complex medium to the optical densities indicated above the gels. Primers used for RNA probe generation are listed in Table S2 in the supplemental material. Ethidium bromide-stained 23S RNAs or tRNAs were used as loading controls. nt, nucleotides. (B) Stability of AbcR1. WT and Δhfq mutant cells were grown to an OD₆₀₀ of 1.0 in YEB medium. Samples were taken before (0) and 1, 5, and 10 min after the addition of rifampin (250 μg/ml). Amounts of 8 μg of total RNA were separated on a 10% polyacrylamide gel containing 7 M urea and were detected by Northern analysis using a DNA probe (generated by primers DNAprobe_C2A_fw and DNAprobe_C2A_rv). The size of AbcR1 is given on the right. Ethidium bromide-stained tRNAs were used as loading controls. (C) Relative amounts of AbcR1 in A. tumefaciens WT and the hfq mutant after the addition of rifampin. Northern blot signals (B) were quantified using AlphaEaseFC software.

Fig 3

Altered protein levels in the hfq deletion mutant. (A) Total protein samples from different growth phases (indicated above the gels) of A. tumefaciens wild type (WT), the Δhfq strain, the complemented mutant (Δhfq+hfq), and control strains containing the empty vector (v) (WT+v and Δhfq+v) were loaded on a 12% SDS–PAGE gel. The positions of marker proteins are given on the left in kDa. Eight prominent protein bands accumulating in the mutant were analyzed via MALDI-TOF. The proteins identified with the highest confidence are given on the right. ABC transporter components are underlined. (B) Identification of overexpressed proteins (A) in the Δhfq mutant by MALDI-TOF. Proteins that were ranked at the first position after MALDI-TOF analysis are in boldface and marked by asterisks. All other listed proteins were additionally identified during mass spectrometry.

Fig 4

Altered RNA levels in the hfq deletion mutant. Northern blots determining RNA levels of some of the proteins identified by SDS-PAGE in Fig. 3A. Hybridizations were performed with amounts of 8 μg of total RNA from A. tumefaciens wild type (WT) and the Δhfq mutant grown to the optical densities indicated above. Primers used for RNA probe generation are listed in Table S2 in the supplemental material. Ethidium bromide-stained 23S RNAs were used as loading controls.

Fig 5

Growth defect and altered cell morphology of the Δhfq strain. (A) A. tumefaciens wild type (WT), the Δhfq mutant, the complemented mutant (Δhfq+hfq), and corresponding control strains harboring the empty vector (v) (WT+v and Δhfq+v) were grown in YEB complex medium, and OD₆₀₀ values were plotted over time. Three independent experiments gave comparable results. (B) Microscopic images were taken of A. tumefaciens strains from cultures grown in YEB medium to optical densities indicated above the images. Y-shaped cells are indicated by arrows.

Fig 6

Virulence defect of the A. tumefaciens Δhfq strain on potato discs. Quantitative tumor formation assay on potato tuber discs. (A) Amounts of 10⁴ and 10⁶ cells/disc of A. tumefaciens wild type (WT), the Δhfq mutant, the complemented mutant (Δhfq+hfq), and control strains harboring the empty vector (v) (WT+v and Δhfq+v) were examined for their tumor formation efficiency on potato discs. Tumorigenesis efficiency is scored by the number of tumors per disc (mean value calculated from results of 60 potato tuber discs for each strain in each independent experiment; error bars show standard errors of the means). (B) Representative pictures of tumors on potato discs. Two independent experiments gave similar results.

Fig 7

Presence of T4SS proteins in the WT and hfq mutant. Cells were grown under non-virulence-inducing (− AS) or virulence-inducing conditions (+ AS [1 mM]) in AB minimal medium (AS, acetosyringone). Cell lysates were subjected to SDS-PAGE, followed by Western blotting and detection with specific antisera.

Fig 8

Current model of Hfq-dependent processes in A. tumefaciens. A schematic A. tumefaciens cell is depicted with inner and outer membrane (IM and OM, respectively). mRNAs influenced by Hfq mostly code for periplasmic proteins. Hfq-mediated regulation is exerted via the sRNA AbcR1 (69) (continuous lines) or via yet-to-be-determined sRNAs (dashed lines).