High-resolution time series of Pseudomonas aeruginosa gene expression and rhamnolipid secretion through growth curve synchronization

BMC Microbiology

Methodology Article

João B Xavier¹  Dave van Ditmarsch¹ 

[1] Computational Biology Program, Memorial Sloan-Kettering Cancer Center, 408 East 69 Street, 0021-5604, New York, NY, USA;

关键词: Growth Curve;    Inoculum Density;    Maximum Specific Growth Rate;    Initial Cell Density;    Endpoint Measurement;   

DOI  :  10.1186/1471-2180-11-140

 received in 2011-02-18, accepted in 2011-06-17,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundOnline spectrophotometric measurements allow monitoring dynamic biological processes with high-time resolution. Contrastingly, numerous other methods require laborious treatment of samples and can only be carried out offline. Integrating both types of measurement would allow analyzing biological processes more comprehensively. A typical example of this problem is acquiring quantitative data on rhamnolipid secretion by the opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa cell growth can be measured by optical density (OD600) and gene expression can be measured using reporter fusions with a fluorescent protein, allowing high time resolution monitoring. However, measuring the secreted rhamnolipid biosurfactants requires laborious sample processing, which makes this an offline measurement.ResultsHere, we propose a method to integrate growth curve data with endpoint measurements of secreted metabolites that is inspired by a model of exponential cell growth. If serial diluting an inoculum gives reproducible time series shifted in time, then time series of endpoint measurements can be reconstructed using calculated time shifts between dilutions. We illustrate the method using measured rhamnolipid secretion by P. aeruginosa as endpoint measurements and we integrate these measurements with high-resolution growth curves measured by OD600 and expression of rhamnolipid synthesis genes monitored using a reporter fusion. Two-fold serial dilution allowed integrating rhamnolipid measurements at a ~0.4 h-1 frequency with high-time resolved data measured at a 6 h-1 frequency. We show how this simple method can be used in combination with mutants lacking specific genes in the rhamnolipid synthesis or quorum sensing regulation to acquire rich dynamic data on P. aeruginosa virulence regulation. Additionally, the linear relation between the ratio of inocula and the time-shift between curves produces high-precision measurements of maximum specific growth rates, which were determined with a precision of ~5.4%.ConclusionsGrowth curve synchronization allows integration of rich time-resolved data with endpoint measurements to produce time-resolved quantitative measurements. Such data can be valuable to unveil the dynamic regulation of virulence in P. aeruginosa. More generally, growth curve synchronization can be applied to many biological systems thus helping to overcome a key obstacle in dynamic regulation: the scarceness of quantitative time-resolved data.

【 授权许可】

CC BY   
© van Ditmarsch and Xavier; licensee BioMed Central Ltd. 2011