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

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.

Influence of the ferric uptake regulator (Fur) protein on pathogenicity in Pectobacterium carotovorum subsp. brasiliense.

Iron is an important nutrient for the survival and growth of many organisms. In order to survive, iron uptake from the environment must be strictly regulated and maintained to avoid iron toxicity. The ferric uptake regulator protein (Fur) regulates genes involved in iron homeostasis in many bacteria, including phytopathogens. However, to date, the role played by Fur in the biology of Pectobacterium carotovorum subsp. brasiliense (Pcb1692), an important pathogen of potatoes, has not yet been studied. To this end, we used the lambda recombineering method to generate a fur mutant strain of Pcb1692 and assessed the virulence and fitness of the mutant strain. The results showed that production of siderophores in Pcb1692Δfur increased compared to the Pcb1692 wild-type and the complemented strain Pcb1692Δfur-pfur. However, production of N-acyl homoserine lactone (AHLs), biofilm formation, exopolysaccharide (EPS) production, virulence on potato tubers and swimming motility, were all significantly decreased in Pcb1692Δfur compared to the wild-type and complemented Pcb1692Δfur-pfur strains. The Pcb1692Δfur mutant also demonstrated significant sensitivity to oxidative stress when exposed to H2O2. Consistent with phenotypic results, qRT-PCR results demonstrated that Fur down-regulates genes which encode proteins associated with: iron uptake (HasA-extracellular heme-binding protein and Ferrodoxin-AED-0004132), stress response (SodC-superoxide dismutase), plant cell wall degrading enzymes (PrtA and CelV) and motility (FlhC and MotA). We conclude that the ferric uptake regulator protein (Fur) of Pcb1692 regulates traits that are important to host-pathogens interactions.

A quorum sensing-defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems.

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post-harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall-degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild-type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild-type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild-type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI-inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild-type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down-regulated in the mutant compared with the wild-type strain.