SummaryWe have investigated the role of LapF, one of the two largest proteins encoded in the genome of Pseudomonas putida KT2440, in bacterial colonization of solid surfaces. LapF is 6310 amino acids long, and is localized on the cell surface. The C-terminal region of the protein is essential for its secretion, which presumably requires the ABC transporter encoded by an operon (lapHIJ) adjacent to the lapF gene. Although the initial attachment stages are not different between the wild type and a lapF mutant, microcolony formation and subsequent development of a mature biofilm is impaired in the mutant. This is consistent with the expression pattern of lapF; activation of its promoter takes place at late stages of growth and is regulated by the alternative sigma factor RpoS. A lapF mutant is also affected in individual and competitive plant root colonization. In these assays, mixed microcolonies formed by cells of both the wild-type and the mutant strains could be observed but microcolonies of the mutant alone were not found. These data and the localization of the protein at discrete spots in areas of contact between cells in biofilms suggest that LapF determines the establishment of cell-cell interactions during sessile growth.

LapA and LapF are large extracellular proteins that play a relevant role in biofilm formation by Pseudomonas putida. Current evidence favors a sequential model in which LapA is first required for the initial adhesion of individual bacteria to a surface, while LapF participates in later stages of biofilm development. In agreement with this model, lapF transcription was previously shown to take place at late times of growth and to respond to the stationary-phase sigma factor RpoS. We have now analyzed the transcription pattern of lapA and other regulatory elements that influence expression of both genes. The lapA promoter shows a transient peak of activation early during growth, with a second increase in stationary phase that is independent of RpoS. The same pattern is observed in biofilms although expression is not uniform in the population. Both lapA and lapF are under the control of the two-component regulatory system GacS/GacA, and their transcription also responds to the intracellular levels of the second messenger cyclic diguanylate (c-di-GMP), although in surprisingly reverse ways. Whereas expression from the lapA promoter increases with high levels of c-di-GMP, the opposite is true for lapF. The transcriptional regulator FleQ is required for the modulation of lapA expression by c-di-GMP but has a minor influence on lapF. This work represents a further step in our understanding of the regulatory interactions controlling biofilm formation in P. putida.

The study of the molecular basis of tree diseases is lately receiving a renewed attention, especially with the emerging perception that pathogens require specific pathogenicity and virulence factors to successfully colonize woody hosts. Pathosystems involving woody plants are notoriously difficult to study, although the use of model bacterial strains together with genetically homogeneous micropropagated plant material is providing a significant impetus to our understanding of the molecular determinants leading to disease. The gammaproteobacterium Pseudomonas savastanoi belongs to the intensively studied Pseudomonas syringae complex, and includes three pathogenic lineages causing tumorous overgrowths (knots) in diverse economically relevant trees and shrubs. As it occurs with many other bacteria, pathogenicity of P. savastanoi is dependent on a type III secretion system, which is accompanied by a core set of at least 20 effector genes shared among strains isolated from olive, oleander, and ash. The induction of knots of wild-type size requires that the pathogen maintains adequate levels of diverse metabolites, including the phytohormones indole-3-acetic acid and cytokinins, as well as cyclic-di-GMP, some of which can also regulate the expression of other pathogenicity and virulence genes and participate in bacterial competitiveness. In a remarkable example of social networking, quorum sensing molecules allow for the communication among P. savastanoi and other members of the knot microbiome, while at the same time are essential for tumor formation. Additionally, a distinguishing feature of bacteria from the P. syringae complex isolated from woody organs is the possession of a 15 kb genomic island (WHOP) carrying four operons and three other genes involved in degradation of phenolic compounds. Two of these operons mediate the catabolism of anthranilate and catechol and, together with another operon, are required for the induction of full-size tumors in woody hosts, but not in non-woody micropropagated plants. The use of transposon mutagenesis also uncovered a treasure trove of additional P. savastanoi genes affecting virulence and participating in diverse bacterial processes. Although there is still much to be learned on what makes a bacterium a successful pathogen of trees, we are already untying the knots.