Analysis of N-acylhomoserine lactone dynamics in continuous cultures of Pseudomonas putida IsoF by use of ELISA and UHPLC/qTOF-MS-derived measurements and mathematical models.

In this interdisciplinary approach, the dynamics of production and degradation of the quorum sensing signal 3-oxo-decanoylhomoserine lactone were studied for continuous cultures of Pseudomonas putida IsoF. The signal concentrations were quantified over time by use of monoclonal antibodies and ELISA. The results were verified by use of ultra-high-performance liquid chromatography. By use of a mathematical model we derived quantitative values for non-induced and induced signal production rate per cell. It is worthy of note that we found rather constant values for different rates of dilution in the chemostat, and the values seemed close to those reported for batch cultures. Thus, the quorum-sensing system in P. putida IsoF is remarkably stable under different environmental conditions. In all chemostat experiments, the signal concentration decreased strongly after a peak, because emerging lactonase activity led to a lower concentration under steady-state conditions. This lactonase activity probably is quorum sensing-regulated. The potential ecological implication of such unique regulation is discussed.

Detection of quorum sensing molecules in Burkholderia cepacia culture supernatants with enzyme-linked immunosorbent assays.

The Burkholderia cepacia complex (Bcc) employs a quorum sensing (QS) mechanism which is a cell density-dependent bacterial communication system to regulate certain gene expressions. As with many other Gram-negative bacteria, Burkholderia cepacia species use (N-acyl-)homoserine lactones (AHLs or HSLs) as signalling molecules. Because of the essential role of QS in bacterial behavior, the aim of this study was to demonstrate the applicability of our in-house-developed enzyme-linked immunosorbent assays (ELISAs) for the detection of bacterial activities via HSLs in B. cepacia strain LA3 culture supernatants. For this purpose the previously developed monoclonal antibodies (mAbs) HSL1/2-2C10 and HSL1/2-4H5 were exploited. N-3-Oxo-decanoyl-L-homoserine lactone (3-oxo-C10-HSL) was used as main analyte throughout all experiments. With the bacterial culture medium (named ABC medium) a matrix effect in both ELISAs was visible (slight increase in optical density, shift in test midpoints (IC(50)) and working ranges). For example, ELISA with mAb HSL1/2-2C10 and enzyme tracer HSL3-HRP (HSL derivative conjugated to horseradish peroxidase) had an IC(50) of 120 µg L(-1) for 3-oxo-C10-HSL in phosphate-buffered saline versus 372 µg L(-1) in ABC medium. A significant increase of HSLs in B. cepacia strain LA3 culture supernatants after 12 h to 48 h of growth was observed. Although the analytical result of these immunoassays cannot distinguish HSLs from homoserines (HSs), the appearance of these compounds can be easily followed. Hydrolysis and spiking experiments were carried out with these biological samples. According to our knowledge, these are the first immunoassays for the detection of quorum sensing molecules in biological culture supernatants. This study provides a cost-effective, fast, and sensitive analytical method for detection of HSLs/HSs in biological samples without complex sample preparation and will offer a quick idea about B. cepacia activities. The low sample amount requirement (less than 1 mL) constitutes a tremendous advantage for many analytical questions with biological samples.

Root colonization by Pseudomonas sp. DSMZ 13134 and impact on the indigenous rhizosphere bacterial community of barley.

Over the last few decades, the ability of rhizosphere bacteria to promote plant growth has been considered to be of scientific, ecological, and economic interest. The properties and mechanisms of interaction of these root-colonizing bacteria have been extensively investigated, and plant protection agents that are based on these bacterial strains have been developed for agricultural applications. In the present study, the root colonization of barley by Pseudomonas sp. DSMZ 13134, that is contained in the commercially available plant protection agent Proradix, was examined using the fluorescence in situ hybridization method with oligonucleotide probes and specific gfp-tagging of the inoculant strain in combination with confocal laser scanning microscopy. In the first phase of root colonization, the inoculant strain competed successfully with seed and soil-borne bacteria (including Pseudomonads) for the colonization of the rhizoplane. Pseudomonas sp. DSMZ 13134 could be detected in all parts of the roots, although it did not belong to the dominant members of the root-associated bacterial community. Gfp-tagged cells were localized particularly in the root hair zone, and high cell densities were apparent on the root hair surface. To investigate the impact of the application of Proradix on the structure of the dominant root-associated bacterial community of barley, T-RFLP analyses were performed. Only a transient community effect was found until 3 weeks post-application.

Dynamic regulation of N-acyl-homoserine lactone production and degradation in Pseudomonas putida IsoF.

The biocontrol strain Pseudomonas putida IsoF, which was isolated from a tomato rhizosphere, is a known N-acyl-homoserine lactone (AHL) producer with only one LuxI/LuxR-like quorum-sensing (QS) system. The production and degradation of AHLs were analysed in different growth phases of the bacterium. Using the analytical tools of ultra performance liquid chromatography and high resolution MS, it was possible to determine not only the various AHLs synthesized over time but also their degradation products. 3-oxo-decanoyl-homoserine lactone was found to be the dominant AHL, which reached its maximum in the early logarithmic growth phase. Although the pH of the medium was neutral, the AHLs were degraded thereafter rapidly to the corresponding homoserines and other metabolites. The proposed lactonase gene of P. putida IsoF could not be identified, because it is apparently quite different from hitherto described lactonases. The analytical data were used to calculate the rates and thresholds of AHL production by mathematical modelling, allowing quantitative predictions and a further understanding of the QS-based regulations in this bacterium. This study, combining microbiological, chemical and mathematical approaches, suggests that AHL degradation is an integral part of the whole autoinducer circuit of P. putida IsoF.