The induction of hrp genes

in bacteria occurs soon after the first contact with plant tissue. Expression of hrp genes are detected as early as 1 h after inoculation and continue Alpelisib datasheet to increase for at least 6 h [6]. However, no specific plant-derivatives have been identified as inducers of hrp genes, and in Ralstonia solanacearum some evidence suggests that the full induction of hrp genes requires contact with plant tissues [7]. The hrp genes are also induced in vitro when bacteria are grown in minimal medium with carbon sources such as sucrose, fructose or mannitol, low pH and a low N/C ratio [6]. Minimal media with these characteristics seems to mimic some of the conditions bacteria might find 4EGI-1 order within the apoplast. It has been suggested that the induction of hrp genes after contact with plant tissues could result from alterations in the nutritional status of the bacteria [2, 6]. During the interaction with their host, it is thought that bacteria commonly detect specific plant metabolites, which are used as signals for changing their gene expression patterns, allowing them to adapt to the plant environment. Specific plant molecules such as phenolic β-glycosides, shikimic and quinic acids, and pectin oligomers

have been reported to activate the expression of genes involved in toxin synthesis and cell wall degradation [8–10].

In this study, we used microarray analysis to identify genes of P. syringae pv. phaseolicola check details NPS3121 differentially expressed in response to metabolites present in plant tissue extracts [11]. Bacteria were grown on minimal medium supplemented with bean leaf extract, apoplastic fluid or bean pod extract. By using these three types of extract, we were able to identify check bacterial genes that possibly facilitate the colonization of susceptible plant tissues, such as bean leaves and/or apoplastic fluid which are known targets during the infection process of P. syringae pv. phaseolicola NPS3121 [11, 12]. Results and Discussion Leaf extracts and apoplastic fluid produce highly similar transcriptional responses We decided to test bean leaf and pod extracts and apoplastic fluid since these are thought to be the primary environments that P. syringae pv. phaseolicola encounters during infection, and in which nutrient assimilation, plant signal recognition and stress responses can occur [13, 14, 1, 12]. To this end, P. syringae pv. phaseolicola NPS3121 was grown at 18°C in M9 minimal medium with glucose as a carbon source. When cultures reached the mid-log phase (OD600 nm 0.6) bean leaf extract, apoplastic fluid or bean pod extracts were added to a final concentration of 2% and an equal amount of minimal medium was added to a control culture.

maltophilia. The elucidation of molecular mechanisms underlying these phenotypic differences might be relevant to the identification of new targets for designing rational and effective methods to combat and eradicate S. maltophilia infection. Methods Bacterial isolates and growth conditions Overall, 98 S. maltophilia isolates were investigated: 41 strains collected

from the sputa of CF Selleck YH25448 patients attending the CF Unit at “”Bambino Gesù”" Children’s Hospital and Research Institute of Rome; TEW-7197 ic50 47 strains collected from different sites (30 from respiratory tract, 10 from blood, and 7 from swabs) in non-CF patients attending “”Bambino Gesù”" Children Hospital of Rome, or “”Spirito Santo”" Hospital of Pe scara; and 10 strains (ENV) isolated in Czech Republic from several environmental sources (paddy, soil, rhizosphere tuberous roots, and waste water). Since in severely ill chronic obstructive pulmonary disease (COPD) patients P. aeruginosa clones similar to those in CF persists [52], patients with COPD were not enrolled AZD6094 cell line in the present study. All clinical isolates represented non-consecutive strains isolated from different patients, except for 2 CF patients with 7 and 3 isolates, respectively. The isolates were identified as S. maltophilia by biochemical tests using manual (API 20-NE System; BioMérieux, Marcy-L’Etoile,

France) or automated (Vitek; BioMérieux) systems, then stored at -80°C until use when they were grown at 37°C (and also at 25°C, in the case of ENV strains) in Trypticase Soy broth

(TSB; Oxoid SpA; Garbagnate M.se, Milan, Italy) or Mueller-Hinton agar (MHA; Oxoid) plates unless otherwise noted. Genetic relatedness by PFGE and cluster analysis After digestion of DNA with the Suplatast tosilate restriction enzyme XbaI as previously described [24, 27, 28], PFGE was carried out as follows: initial switch time and final switch time were 5 and 35 sec, respectively; DNA fragments were run with a temperature of 12°C for 20 h at 6.0 V/cm with an included angle of 120°. Isolates with identical PFGE patterns were assigned to the same PFGE type and subtype. Isolates differing by one to three bands were assigned to different PFGE subtypes but to the same PFGE type and were considered genetically related. Isolates with PFGE patterns differing by more than 4 bands were considered genetically unrelated and were assigned to different PFGE types. PFGE types were analyzed with BioNumerics software for Windows (version 2.5; Applied Maths, Ghent, Belgium). The DNA banding patterns were normalized with bacteriophage lambda concatemer ladder standards. Comparison of the banding patterns was performed by the UPGMA and with the Dice similarity coefficient. A tolerance of 1.