Strains Used In This Study All strains of S. typhimurium are derived from Sl1344. Introduction ! Salmonella enterica serovar Typhimurium alternates expression of two antigenically distinct flagellins that form the flagellar filament ! Flagellar phase variation was first discovered in 1922 (1) but the biological significance and signals controlling flagellin switching are poorly understood ! Alternate flagellin expression is caused by a well-studied DNA inversion mechanism catalyzed by the Hin recombinase (2): ! Although Hin levels control the rate of flagellin switching, there is no obvious reason for the inversion reaction to favour one orientation over the other ! However, FliC is the predominant flagellin in wild-type cells ! We have previously shown that RdoA expression influences the bias towards FliC expression (3) ! RdoA is a eukaryotic-like kinase (4) that is upregulated via the Cpx two component signal transduction pathway (5): Our hypothesis is that RdoA activity helps to maintain the bias toward FliC expression. To explore this hypothesis, the following questions were asked: ! How does RdoA affect FljB expression? ! Does RdoA affect the orientation of the invertible segment involved in flagellar phase variation? ! Does RdoA affect Hin expression? ! How do different conditions activate the Cpx pathway? ! Does RdoA affect overall flagellation or flagellar function? Does RdoA affect the functionality of flagella or overall flagellation? ! Used a swimming motility assay to determine ability of wild-type and rdoA strains to be motile ! Used method of Turner et al. (6) to stain flagella of wild-type and rdoA cells using Alexa fluor 546 to observe overall flagellation Wild-type rdoA ! Wild-type appears to have less abundant flagella than rdoA ! RdoA also binds more dye to cell body than wild-type ! Could be due to: - higher affinity of Alexa 546 for FljB - flagella more ‘bundled’ in wild-type ! Suggests that: - RdoA maintains lower level of flagellation - increased abundance of flagella in rdoA due to heightened FljB expression ! There is no difference in swimming motility in the absence of RdoA - suggests RdoA does not affect the functionality of flagella Discussion RdoA normally maintains cells in a FliC-expressing phase. ! RdoA maintains lower FljB expression ! RdoA maintains the invertible segment in a FliC-ON orientation The absence of RdoA, and alkaline pH, activate the Cpx pathway. ! Both of these conditions also cause a decrease in Hin expression ! Decrease in Hin expression is likely due to Cpx activation, not absence of RdoA ! Therefore, RdoA does not elicit changes in flagellin expression bias by changing Hin levels RdoA maintains a lower level of flagellation but does not affect function of flagella. ! More abundant flagella in the absence of RdoA ! No change in swimming motility in the absence of RdoA ! Perhaps increased FljB in absence of RdoA is an increase in overall number of flagella by increasing FljB-containing flagella more than FliC-containing flagella. Taken together, these results strongly support our hypothesis that RdoA maintains the bias towards FliC expression. Our results also suggest that this effect occurs through alteration of the orientation of the invertible segment involved in phase variation and through changes in overall flagellin expression. The effect of RdoA is not through its actions on Hin expression. Why is RdoA regulated by the Cpx pathway? ! To assist in regulation of surface appendage expression ! To optimize this expression when signals of environmental change are detected, as in infection ! To coordinately regulate flagellar expression with other Cpx-regulated proteins RdoA Do RdoA or pH affect FljB expression? ! We wanted to see how RdoA and Cpx activation (pH 8.5) affects the proportion of cells expressing fljB ! Measured fljB promoter activity in a chromosomal fljB::lacZ transcriptional reporter fusion ! Therefore, are assessing phase variation-dependent FljB expression at a PER-CELL level ! Twice as many cells express FljB in the absence of RdoA ! Therefore, RdoA normally causes more cells to be in the FliC- expressing phase ! pH 8.5 data is not significantly different from pH 7 data (not shown) ! Suggests Cpx activation has little or no influence on flagellin expression, compared to RdoA Does RdoA affect phase variation through DNA inversion? ! We wanted to see if RdoA affects the orientation of the invertible segment responsible for flagellar phase variation on a POPULATION basis ! Tested using PCR reactions with primers that produce different sized amplicons depending on the orientation of the segment: ! FljB-expressing orientation: 430 bp amplicon ! FliC-expressing orientation: 200 bp amplicon ! control amplicon for entire region: 1066 bp (same size regardless of orientation, used to control for amount of DNA) Control FljB-ON FliC-ON Wild-type rdoA ! In wild-type, FliC-expressing orientation predominates ! In rdoA, FljB-expressing orientation predominates ! Therefore, RdoA normally maintains cells in the FliC- expressing phase ! Activating Cpx using pH 8.5, or using cpxAR, cpxR or rdoAcpxR strains, FliC-expressing orientation predominates, as in wild-type (data not shown) ! Therefore, the effect on DNA inversion occurs predominantly through RdoA The Role of RdoA in flagellar phase variation in Salmonella enterica Richards, M. S., Manu-Boateng, A., & Martin, N. L. Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario, Canada K7L 3N6 fliC fliC fljA fljA fljB fljB promoter promoter OFF OFF ON ON DNA inversion, catalyzed by Hin recombinase P fliC P fliC FljB FliC FljA 996 bp CpxA H~ P H CpxA misfolded periplasmic proteins NlpE overexpression pH 8.5 environment ATP ADP CpxR-P CpxR P i periplasmic foldases CpxP RdoA CpxP Periplasm Cytoplasm Cpx signaling pathway References 1. Andrewes, F. W. (1922) Studies in group-agglutination. I. The Salmonella group and its antigenic structure. J Pathol Bacteriol. 25: 505-521. 2. Bruist, M. F. and Simon, M. I. (1984) Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification. J Bacteriol. 159: 71-79. 3. Suntharalingam, P., Spencer, H., Gallant, C. V. and Martin, N. L. (2003) Salmonella enterica serovar Typhimurium rdoA is growth phase regulated and involved in relaying cpx- induced signals. J Bacteriol. 185: 432-443. 4. He, C. (2005) Master’s Thesis. Queen’s University. 5. Raivio, T. L. and Silhavy, T. J. (1997) Transduction of envelope stress in Escherichia coli by the Cpx two-component system. J Bacteriol. 179: 7724-7733. 6. Turner, L., Ryu, W. S. and Berg, H. C. (2000) Real time imaging of fluorescent flagellar filaments. J Bacteriol. 182: 2793-2801. Does activating the Cpx pathway influence Hin levels? ! Because of the changes in Hin levels we saw, we next wanted to know if these changes may be due to the activation of the Cpx pathway ! Used a cpxP::lacZ transcriptional reporter fusion in wild-type and rdoA cells grown at pH 7.0 or 8.5 to measure Cpx activation using a B-galactosidase assay ! In wild-type, the Cpx pathway is induced by growth at pH 8.5 ! The Cpx pathway is also induced by the absence of RdoA ! Suggests that changes in Hin expression are due to Cpx activation, NOT due to effects of RdoA directly Results Does RdoA affect Hin expression? ! We next tried to determine if the target of RdoA’s actions with regard to phase variation are due to effects on Hin ! Used a hin::lacZ translational reporter fusion in wild-type and rdoA cells grown at pH 7.0 or 8.5 to measure Hin expression using a B-galactosidase assay ! In wild-type, Hin expression is higher than in rdoA at both pHs ! pH 8.5 also decreases Hin expression ! Results suggest that Hin levels in all growth conditions are sufficient to promote phase variation ! Is the effect on Hin due to RdoA, or due to Cpx activation? Acknowledgements Funding provided to M.S.R. via the Digestive Sciences CIHR Training Grant at Queen’s University and NSERC. Funding provided to A.M.B. via the Dean’s Canadian Visible Minority Award. Operating grant support from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR) is gratefully acknowledged. fliC fliC fljA fljA fljB fljB promoter promoter OFF OFF ON ON DNA inversion, catalyzed by Hin recombinase P fliC P fliC FljB FliC FljA 996 bp Future Directions ! To determine HOW RdoA maintains the bias toward FliC expression (i.e. Through another protein, or directly?) ! To determine the targets of RdoA’s kinase activity ! To determine if Cpx and RdoA effects occur in conjunction with one another, or through separate routes Although the mechanism underlying flagellar phase variation in S. typhimurium is well understood, there is still much to be learned about why phase variation occurs and how it is stimulated. Determining the role of RdoA in flagellar phase variation will help to link this process with cell physiology and environment. 1 um 1 um