Strains Used In This StudyAll strains of S typhimurium are derived from Sl1344

IntroductionSalmonella enterica serovar Typhimurium alternates expression of two antigenically distinct flagellins that form the flagellar filamentFlagellar phase variation was first discovered in 1922 (1) but the biological significance and signals controlling flagellin switching are poorly understoodAlternate 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 otherHowever FliC is the predominant flagellin in wild-type cellsWe 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 askedHow does RdoA affect FljB expressionDoes RdoA affect the orientation of the invertible segment involved in flagellar phase variationDoes RdoA affect Hin expressionHow do different conditions activate the Cpx pathwayDoes 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 motileUsed 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 rdoAWild-type appears to have less abundant flagella than rdoARdoA also binds more dye to cell body than wild-typeCould be due to- higher affinity of Alexa 546 for FljB- flagella more lsquobundledrsquo in wild-typeSuggests that- RdoA maintains lower level of flagellation- increased abundance of flagella in rdoA due to heightened FljB expressionThere is no difference in swimming motility in the absence of RdoA- suggests RdoA does not affect the functionality of flagella

DiscussionRdoA normally maintains cells in a FliC-expressing phaseRdoA maintains lower FljB expressionRdoA maintains the invertible segment in a FliC-ON orientationThe absence of RdoA and alkaline pH activate the Cpx pathwayBoth of these conditions also cause a decrease in Hin expressionDecrease in Hin expression is likely due to Cpx activation not absence of RdoATherefore RdoA does not elicit changes in flagellin expression bias by changing Hin levelsRdoA maintains a lower level of flagellation but does not affect function of flagellaMore abundant flagella in the absence of RdoANo change in swimming motility in the absence of RdoAPerhaps increased FljB in absence of RdoA is an increase in overall number of flagella by increasing FljB-containing flagella more than FliC-containing flagellaTaken 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 expressionWhy is RdoA regulated by the Cpx pathwayTo assist in regulation of surface appendage expressionTo optimize this expression when signals of environmental change are detected as in infectionTo 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 85) affects the proportion of cells expressing fljB Measured fljB promoter activity in a chromosomal fljBlacZ 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 85 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 85 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 entericaRichards M S Manu-Boateng A amp Martin N LDepartment of Microbiology and Immunology Queenrsquos University Kingston Ontario Canada K7L 3N6

fliC

fliC

fljA

fljAfljB

fljBpromoter

promoterOFF

OFFON

ON

DNA inversion catalyzedby Hin recombinase

PfliC

PfliC

FljB

FliC

FljA

996 bp

CpxA

H~ PH

CpxA

misfoldedperiplasmic

proteinsNlpE

overexpressionpH 85

environment

ATP ADP

CpxR-P

CpxR

Pi

periplasmicfoldases

CpxPRdoA

CpxPPeriplasmCytoplasm

Cpx signalingpathway

References1 Andrewes F W (1922) Studies in group-agglutination I The Salmonella group and its antigenic structure J Pathol Bacteriol 25 505-5212 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 71-793 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 185 432-4434 He C (2005) Masterrsquos Thesis Queenrsquos 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 7724-77336 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 levelsBecause 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 pathwayUsed a cpxPlacZ transcriptional reporter fusion in wild-type and rdoA cells grown at pH 70 or 85 to measure Cpx activation using a B-galactosidase assay

In wild-type the Cpx pathway is induced by growth at pH 85The Cpx pathway is also induced by the absence of RdoASuggests 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 RdoArsquos actions with regard to phase variation are due to effects on Hin Used a hinlacZ translational reporter fusion in wild-type and rdoA cells grown at pH 70 or 85 to measure Hin expression using a B-galactosidase assay

In wild-type Hin expression is higher than in rdoA at both pHs pH 85 also decreases Hin expression Results suggest that Hin levels in all growth conditions are sufficient to promote phase variationIs the effect on Hin due to RdoA or due to Cpx activation

AcknowledgementsFunding provided to MSR via the Digestive Sciences CIHR Training Grant at Queenrsquos University and NSERCFunding provided to AMB via the Deanrsquos Canadian Visible Minority AwardOperating 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

fljAfljB

fljBpromoter

promoterOFF

OFFON

ON

DNA inversion catalyzedby Hin recombinase

PfliC

PfliC

FljB

FliC

FljA

996 bp

Future DirectionsTo determine HOW RdoA maintains the bias toward FliC expression (ie Through another protein or directly)To determine the targets of RdoArsquos kinase activityTo determine if Cpx and RdoA effects occur in conjunction with one another or through separate routesAlthough 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 stimulatedDetermining the role of RdoA in flagellar phase variation will help to link this process with cell physiology and environment

1 um 1 um