Pectobacterium brasiliense 1692 Chemotactic Responses and the Role of Methyl-Accepting Chemotactic Proteins in Ecological Fitness

Abstract

To adapt to changing environmental niches, bacteria require taxis, a movement toward or away from a stimulus (ligand). Chemotaxis has been studied in some members of the Soft Rot Pectobacteriaceae (SRP), particularly members of the genus Dickeya. On the contrary, there are fewer studies on this topic for the other genus in the SRP group, namely Pectobacterium. This study evaluated chemotactic responses in Pectobacterium brasiliense (Pb 1692) to various ligands. A total of 34 methyl-accepting chemotactic proteins (MCPs) were identified in the Pb 1692 genome and the domain architectures of these MCPs were determined. Four Pb 1692 MCPs previously shown to be differentially expressed during potato tuber infection were selected for further functional characterization. Toward this end, Pb 1692 mutant strains each lacking either AED-0001492, AED-0003671, AED-0000304, or AED-0000744 were generated. Two of these mutants (AED-0001492 and AED-0003671), were attenuated in their ability to grow and respond to citrate and are thus referred to as MCP _cit2 and MCP _cit1 , respectively, while the other two, AED-0000304 (MCP _xyl ) and AED-0000744 (MCP _asp ), were affected in their ability to respond to xylose and aspartate, respectively. Trans-complementation of the mutant strains restored swimming motility in the presence of respective ligands. The four MCP mutants were not affected in virulence but were significantly attenuated in their ability to attach to potato leaves suggesting that ecological fitness is an important contribution of these MCPs toward Pb 1692 biology.

Keywords: Pectobacterium brasiliense; blackleg disease; chemotaxis; ecological fitness; methyl accepting chemotaxis proteins; soft rot; virulence.

FIGURE 1

Representation of domain organization of all 34 MCP in Pb 1692 strain. The organization containing both MCP and HAMP domains is the most common in Pb 1692 strain. All predicted domain structures were made through HMMER software with the support of the Pfam-A database. Transmembrane regions were predicted by the Phobius algorithm. The genes highlighted: (1) black was not upregulated, (2) blue was down-regulated, and (3) red was up-regulated during in-planta infection. Furthermore, genes with “*” indicated those mutated and characterized.

FIGURE 2

Swimming motility assay. Pb 1692 wild-type was spotted on minimal media supplemented with 1 mM of xylose (A), aspartate (B), and citrate (C,D). Similarly, mutant strains lacking AED-0000304 (Pb1692ΔMCP_xyl), AED-0000744 (Pb1692ΔMCP_asp), AED-0003671 (Pb1692ΔMCP_cit1), and AED-0001492 (Pb1692ΔMCP_cit2) were spotted on minimal media supplemented with 1 mM of xylose (E), aspartate (F) and citrate (G,H), respectively. Complemented strains Pb1692ΔMCP_xyl–pxyl Pb1692ΔMCP_asp–pasp, Pb1692ΔMCP_cit1–pcit1, and Pb1692ΔMCP_cit2–pcit2, were spotted on minimal media supplemented with 1 mM of aspartate (I), xylose (J) citrate (K,L), respectively.

FIGURE 3

MCP mutants were attenuated in their ability to attach to potato leaves. Determination of CFU/ml from potato leaves after 2 h incubation with 10 μl of each mutant strain (OD₆₀₀ equivalent to 1.0) in M9 media. Three independent experiments were performed each with three technical replicates with a corresponding standard error of the mean. Statistically significant differences (p < 0.05) between mutants, wild-type, and complemented strains are shown by an asterisk.