Development of reporter genes for use in Gram positive bacteria
Green fluorescent protein (gfp) and bacterial luminescence (lux) reporter genes have been used to construct a variety of reporter plasmids for Gram positive bacteria with the aim of using these for bacterial localisation and gene expression studies. The native gfp and luxCDABE genes were cloned into a shuttle vector and the resulting plasmids used to transform Listeria monocytogenes. However, the bacterial populations were found to be weakly fluorescent or luminescent compared to E. coli harbouring the same plasmids. When L. monocytogenes expressing gfp were examined by fluorescence microscopy, only a small proportion of the population was seen to fluoresce. This phenomenon was observed regardless of the gfp variant used in the cloning procedure. However, when gfp3 was placed downstream of PxylA, slightly more individual fluorescent cells were observed compared to when gfp3 was expressed from Pxyn, but the majority of the population was still non-fluorescent. Northern blot analysis and subsequent analysis by SDS PAGE and immunoblotting lead to the supposition that translation of gfp was limiting in L. monocytogenes. A variety of factors could potentially lead to poor translation of the protein, for example poor codon usage, the presence of a ribosome stall site, or poor initiation of translation by the ribosomes. These were all investigated in tum to determine why translation of gfp3 was limiting. Modification of the translational initiation region of gfp3, resulted in a homogeneously fluorescent L. monocytogenes population when the modified gene was expressed from PxylA. Individual lux genes, luxA, luxC and luxE were also translationally enhanced in a similar way to gfp3, and reorganised into an operon where the luciferase genes were adjacent to, but separate from the aldehyde genes. This engineered luxABCDE operon was also expressed from PxylA and highly luminescent populations of L. monocytogenes and Staphylococcus aureus obtained. Having optimised translation for expression III Gram positive bacteria, these reporters were used to construct a variety of reporter plasmids that were successfully employed to observe the intracellular invasion and to monitor agr expression in S. aureus.