Edwardsiella ictaluri is a Gram-negative bacterium and the causative agent of enteric septicemia of catfish. In this study, we examined the expression and function of the LuxS from a pathogenic E. ictaluri strain, J901. J901 was found to produce autoinducer 2 (AI-2) activity that maximized at mid-logarithmic phase and was enhanced by glucose and repressed by high temperature. Consistently, a luxS gene (luxSEi) was identified in J901, whose expression was regulated by cell density, glucose, and temperature in a manner similar to that observed with AI-2 activity. Further analysis showed that LuxSEi is a biologically active AI-2 synthase that was able to complement the luxS-defective phenotype of Escherichia coli DH5. To examine the functional importance of LuxSEi, a genetically modified variant of J901, J901Ri, was constructed, in which luxSEi expression was blocked by RNA interference. Compared to the wild type, J901Ri was (i) reduced in AI-2 activity to a level of 59% of that of the wild type; (ii) impaired in both planktonic and biofilm growth; (iii) significantly attenuated in the ability to infect cultured fish cells and to cause mortality in infected fish; (iv) unable to induce the expression of certain virulence-associated genes. Addition of exogenous AI-2 failed to rescue the growth defect of J901Ri as free-living cells but restored biofilm production and the expression of virulence genes to levels comparable to those of the wild type. Taken together, these results indicate that LuxSEi is a functional AI-2 synthase that is required for optimal cellular growth and host infection.
The influence of bacterial activities on inorganic nutrients has always affected total phytoplankton uptake rates owing to the absence of a reliable method that can exclude these effects. The use of natural samples to determine the contribution of bacterial activities has been based on the size fractionation method which, unfortunately, is encumbered with uncertainties, especially because of the size overlap between bacteria and phytoplankton communities. In this paper, the results are reported of an estimation of bacterial activities by the use of inhibitors (antibiotics). It was shown that the contribution of bacterial activities to the uptake of nitrogenous nutrients was highest for ammonium (79%), followed by nitrate (72%) and urea (62%). In a second set of experiments the concentration of ammonium was raised by 5 μM. This was done to avoid nutrient limitation resulting from the absence of recycled nutrients following the addition of antibiotics and the maximum contribution of bacterial activity to the uptake rate of ammonium increased to 87%. It can be concluded that the use of inhibitors is a good method, a reliable alternative to the fractionation method. However, it is important to note that inhibitors can affect both phytoplankton growth and the nutrient recycling process. Our results indicate that the application of antibiotics had measurable effects not only on the target bacteria but also on the uptake behaviour of phytoplankton. Our observations were therefore limited to the period when there was no effect on the phytoplankton, as was demonstrated by a carbon protein incorporation experiment.