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. 2003 Sep 16;100(19):10995-1000.
doi: 10.1073/pnas.1833360100. Epub 2003 Sep 5.

Biofilm dispersal in Xanthomonas campestris is controlled by cell-cell signaling and is required for full virulence to plants

J Maxwell Dow  1 , Lisa Crossman, Kim Findlay, Yong-Qiang He, Jia-Xun Feng, Ji-Liang Tang
Affiliations

Biofilm dispersal in Xanthomonas campestris is controlled by cell-cell signaling and is required for full virulence to plants

J Maxwell Dow et al. Proc Natl Acad Sci U S A. .

Abstract

The rpf gene cluster of Xanthomonas campestris pathovar campestris (Xcc) is required for the pathogenesis of this bacterium to plants. Several rpf genes are involved in the coordinate positive regulation of the production of virulence factors mediated by the small diffusible molecule DSF (for diffusible signal factor). RpfF directs the synthesis of DSF, and a two-component sensory transduction system comprising RpfC and RpfG has been implicated in the perception of the DSF signal and signal transduction. In L medium, rpfF, rpfG, rpfC, and rpfGHC mutants grew as matrix-enclosed aggregates, whereas the wild type grew in a dispersed planktonic fashion. Synthesis of the extracellular polysaccharide xanthan was required for aggregate formation. Addition of DSF triggered dispersion of the aggregates formed by the rpfF strain, but not those of rpf strains defective in DSF signal transduction. An extracellular enzyme from Xcc whose synthesis was positively controlled by the DSF/rpf system could disperse the aggregates produced by all rpf strains. The enzyme was identified as the single endo-beta-1,4-mannanase encoded by the Xcc genome. This enzyme had no detectable activity against soluble xanthan. The endo-beta-1,4-mannanase was required for the full virulence of Xcc to plants. On the basis of this model system, we propose that one role of the beta-mannanase during disease is to promote transitions from an aggregated or biofilm lifestyle to a planktonic lifestyle in response to the DSF signal.

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Figures

Fig. 1.
Fig. 1.
Formation of aggregates by X. campestris and the role of DSF. (A) Strains of X. campestris with mutations in rpfF, rpfG, and rpfC grow in an aggregated fashion in L medium, whereas the wild-type strain (8004) grows in a dispersed fashion. Addition of the DSF to rpfF strains but not to rpfG or rpfC strains causes dispersed growth. (B) Scanning electron microscopy reveals that aggregates of rpf strains have a reticulated structure where bacteria are held within a matrix. (Scale bar = 10 μm.) (C) Wild-type bacteria in contrast show no such matrix encasement. (Scale bar = 5 μm.) Note that these bacteria showed no aggregation in light microscopy before application to the membrane used in scanning electron microscopy.
Fig. 2.
Fig. 2.
An extracellular enzyme from X. campestris can disperse the aggregates. (A) Light microscopy of cultures of an rpf mutant after 24 h of growth. (B) The same culture after treatment for 30 min with the dispersing enzyme. (C) Coomassie blue-stained SDS/PAGE of the most active fraction from ion-exchange chromatography of the dispersing enzyme preparation. Molecular mass standards are given in kilodaltons; protein bands 1and 2 are indicated.
Fig. 3.
Fig. 3.
Synthesis of the endo-β-1,4-mannanase is positively regulated by the rpf genes in NYGB medium. Addition of DSF restores enzyme production to the rpfF mutant. In L medium, enzyme activity was not detected in culture supernatants of rpf mutants.
Fig. 4.
Fig. 4.
The endo-β-1,4-mannanase is required for full virulence of X. campestris to plants. (A) Symptom production on leaves 14 days after inoculation by clipping with (from the left) wild type, two manA mutant strains (derived independently), and water (mock inoculation). (B) The average lesion lengths caused by the manA mutant are significantly shorter (P < 0.01) than those by the wild type at two different inoculation levels (indicated by OD at 600 nm of 0.1 and 0.001). Values are the mean ± SD from 15 measurements. (C) The percentage of diseased plants is also lower after spray inoculation with the manA mutant than with the wild type at two inoculum levels. Results from a typical experiment are shown. This experiment and that shown in B were repeated two more times with essentially the same result. (D) The manA mutant (right leaf) shows reduced spread through the vascular system of infected plants compared with the wild type (left leaf) as demonstrated by staining for bacteria constitutively expressing β-glucuronidase. Infected leaves were stained for β-glucuronidase activity at 4 days after leaf clipping.
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