Rhizosphere selection of Pseudomonas putida KT2440 variants with increased fitness associated to changes in gene expression.

As the interface between plant roots and soil, the rhizosphere is a complex environment where nutrients released by the plant promote microbial growth. Increasing evidences indicate that the plant also exerts a selective pressure on microbial populations in the rhizosphere, favouring colonization by certain groups. In this work, we have designed an experimental setup to begin analysing the evolution of a specific bacterial population in the rhizosphere, using Pseudomonas putida KT2440 as model organism. After several rounds of selection without passage through laboratory growth conditions, derivatives of this strain with increased fitness in the rhizosphere were isolated. Detailed analysis of one of these clones indicated that this effect is specific for rhizosphere conditions and derives from changes in its transcriptional profile in this environment, with 43 genes being differentially expressed with respect to the parental strain. Several of these genes belong to functional categories which could affect stress adaptation and availability of particular nutrients. By inactivating two genes identified as upregulated in the selected clone (coding for a stress-response protein and a rRNA modifying protein), these functions were shown to contribute to rhizosphere fitness. Our data also suggest the existence of different evolutionary pathways leading to increased rhizosphere fitness.

Congenital ulcer of the buttock.

We present the case of a congenital, deep ulcer of the buttock in an otherwise healthy newborn girl. Histopathology excluded any specific cause of ulceration. The ulcer healed spontaneously at 4 weeks of age, leaving a scar. Because the patient also had symmetrical pits on her knees and because the ulcer was located in an area typically affected by dimples, we interpret this unique manifestation as a congenital defect in the spectrum of congenital dimples.

Interplay between extracellular matrix components of Pseudomonas putida biofilms.

The extracellular matrix of bacterial biofilms has at least two key functions: to serve as a structural scaffold for the multicellular community, and to play a protective role against external stress. In this work, we report a compensatory effect whereby Pseudomonas putida reacts to the lack of either of the two main surface proteins involved in biofilm formation, LapA and LapF, by increasing expression and production of a species-specific EPS. Elevated levels of the second messenger molecule cyclic di-GMP alter the balance of extracellular matrix components, and the phenotypes of lapA and lapF mutants under these conditions are indicative of direct interactions taking place between large secreted proteins and exopolysaccharides. Our data suggest the existence of a mechanism by which bacteria would sense alterations in the composition of the extracellular matrix, leading to changes in expression of the different elements.

Stability of a Pseudomonas putida KT2440 bacteriophage-carried genomic island and its impact on rhizosphere fitness.

The stability of seven genomic islands of Pseudomonas putida KT2440 with predicted potential for mobilization was studied in bacterial populations associated with the rhizosphere of corn plants by multiplex PCR. DNA rearrangements were detected for only one of them (GI28), which was lost at high frequency. This genomic island of 39.4 kb, with 53 open reading frames, shows the characteristic organization of genes belonging to tailed phages. We present evidence indicating that it corresponds to the lysogenic state of a functional bacteriophage that we have designated Pspu28. Integrated and rarely excised forms of Pspu28 coexist in KT2440 populations. Pspu28 is self-transmissible, and an excisionase is essential for its removal from the bacterial chromosome. The excised Pspu28 forms a circular element that can integrate into the chromosome at a specific location, att sites containing a 17-bp direct repeat sequence. Excision/insertion of Pspu28 alters the promoter sequence and changes the expression level of PP_1531, which encodes a predicted arsenate reductase. Finally, we show that the presence of Pspu28 in the lysogenic state has a negative effect on bacterial fitness in the rhizosphere under conditions of intraspecific competition, thus explaining why clones having lost this mobile element are recovered from that environment.

Selection of hyperadherent mutants in Pseudomonas putida biofilms.

A number of genetic determinants required for bacterial colonization of solid surfaces and biofilm formation have been identified in different micro-organisms. There are fewer accounts of mutations that favour the transition to a sessile mode of life. Here we report the isolation of random transposon Pseudomonas putida KT2440 mutants showing increased biofilm formation, and the detailed characterization of one of them. This mutant exhibits a complex phenotype, including altered colony morphology, increased production of extracellular polymeric substances and enhanced swarming motility, along with the formation of denser and more complex biofilms than the parental strain. Sequence analysis revealed that the pleiotropic phenotype exhibited by the mutant resulted from the accumulation of two mutations: a transposon insertion, which disrupted a predicted outer membrane lipoprotein, and a point mutation in lapG, a gene involved in the turnover of the large adhesin LapA. The contribution of each alteration to the phenotype and the possibility that prolonged sessile growth results in the selection of hyperadherent mutants are discussed.

PpoR, an orphan LuxR-family protein of Pseudomonas putida KT2440, modulates competitive fitness and surface motility independently of N-acylhomoserine lactones.

Pseudomonas putida KT2440 does not produce any of the common molecules involved in quorum sensing signalling described in other bacteria. However, as is the case in other microorganisms, the genome of this strain contains an open reading frame (PP_4647) coding for a transcriptional regulator belonging to the LuxR protein family. In this article, we present evidence indicating that this protein, named PpoR, modulates swarming motility in KT2440 and plays a role in the survival of this strain in the presence of potential competitors. These functions appear to be independent of known N-acylhomoserine lactones (AHLs), since we show that P. putida KT2440 does not produce significant quantities of these molecules under any condition tested and PpoR does not influence the expression of quorum sensing-dependent promoters even in the presence of exogenous AHLs. A ppoR mutant shows increased sensitivity to the iron chelator 2,2'-dipyridyl, while iron supplementation compensates the fitness loss of the mutant in competition with other Pseudomonas. All these data suggest that PpoR participates in both inter- and intraspecific processes relevant to the fitness of P. putida related to iron acquisition, and not necessarily mediated by canonical quorum sensing signal molecules.