Reconstructing promoter activity from Lux bioluminescent reporters.

The bacterial Lux system is used as a gene expression reporter. It is fast, sensitive and non-destructive, enabling high frequency measurements. Originally developed for bacterial cells, it has also been adapted for eukaryotic cells, and can be used for whole cell biosensors, or in real time with live animals without the need for euthanasia. However, correct interpretation of bioluminescent data is limited: the bioluminescence is different from gene expression because of nonlinear molecular and enzyme dynamics of the Lux system. We have developed a computational approach that, for the first time, allows users of Lux assays to infer gene transcription levels from the light output. This approach is based upon a new mathematical model for Lux activity, that includes the actions of LuxAB, LuxEC and Fre, with improved mechanisms for all reactions, as well as synthesis and turn-over of Lux proteins. The model is calibrated with new experimental data for the LuxAB and Fre reactions from Photorhabdus luminescens-the source of modern Lux reporters-while literature data has been used for LuxEC. Importantly, the data show clear evidence for previously unreported product inhibition for the LuxAB reaction. Model simulations show that predicted bioluminescent profiles can be very different from changes in gene expression, with transient peaks of light output, very similar to light output seen in some experimental data sets. By incorporating the calibrated model into a Bayesian inference scheme, we can reverse engineer promoter activity from the bioluminescence. We show examples where a decrease in bioluminescence would be better interpreted as a switching off of the promoter, or where an increase in bioluminescence would be better interpreted as a longer period of gene expression. This approach could benefit all users of Lux technology.

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria.

BACKGROUND: The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria. RESULTS: Comparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning. CONCLUSION: The residual att sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.

Alternative roles of ClpX and ClpP in Staphylococcus aureus stress tolerance and virulence.

Clp proteolytic complexes are essential for virulence and for survival under stress conditions in several pathogenic bacteria. Recently, a study using signature-tagged mutagenesis identified the ClpX ATPase as also being required for virulence in Staphylococcus aureus. Presently, we have constructed deletion mutants removing either ClpX or the proteolytic subunit, ClpP, in S. aureus 8325-4 in order to examine a putative link between stress tolerance and virulence. When exposed to stress, we found that, although clpP mutant cells were sensitive to conditions generating misfolded proteins, the absence of ClpX improved survival. In the presence of oxidative stress or at low temperature, both ClpP and ClpX were important for growth. Virulence was examined in a murine skin abscess model and was found to be severely attenuated for both mutants. S. aureus pathogenicity is largely dependent on a set of extracellular and cell wall-associated proteins. In the mutant cells, the amount of alpha-haemolysin (hla) and several other extracellular proteins was greatly decreased, and analysis of hla expression revealed that the reduction occurred at the transcriptional level. Essential for transcriptional regulation of hla is the quorum-sensing agr locus. Interestingly, the absence of ClpX or ClpP reduced both transcription of the agr effector molecule, RNA III, and the activity of the autoinducing peptide (AIP). In addition, ClpX was required independently of ClpP for transcription of spa encoding Protein A. Thus, our results indicate that ClpX and ClpP contribute to virulence by controlling the activity of major virulence factors rather than by promoting stress tolerance.