Expression of ica operon-mediated biofilm formation in Staphylococcus epidermidis RP62A is subject to phase variable regulation . Reversible transposition of IS256 into icaADBC or downregulation of icaADBCexpression are two important mechanisms of biofilm phenotypicvariation . Interestingly, the presence of IS256 was generallyassociated with a more rapid rate of phenotypic variation, suggestingthat IS256 insertions outside the ica locus may affect ica transcription.Consistent with this, we identified variants with diminishedica expression, which were associated with IS256 insertionsin the ^B activator rsbU or sarA . Biofilm development and ica expression were activated only by ethanol and not NaCl in rsbU::IS256 insertion variants, which were present in 11% of all variants. ^B activity was impaired in rsbU::IS256 variants, as evidenced by reduced expression of the ^B-regulated genes asp23, csb9,and rsbV . Moreover, expression of sarA, which is ^B regulated,and SarA-regulated RNAIII were also suppressed . A biofilm-formingphenotype was restored to rsbU::IS256 variants only after repeatedpassage and was not associated with IS256 excision from rsbU.Only one sarA::IS256 insertion mutant was identified among 43biofilm-negative variants . Both NaCl and ethanol-activated icaexpression in this sarA::IS256 variant, but only ethanol increasedbiofilm development . Unlike rsbU::IS256 variants, reversionof the sarA::IS256 variant to a biofilm-positive phenotype wasaccompanied by precise excision of IS256 from sarA and restorationof normal ica expression . These data identify new roles forIS256 in ica and biofilm phenotypic variation and demonstratethe capacity of this element to influence the global regulationof transcription in S . epidermidis.

 
Biofilm formation by staphylococci on implanted biomaterialsis now recognized as an important virulence factor contributingto the development of a significant proportion of all device-related infections . Enzymes encoded by the icaADBC operon [20, 23, 27]are required for synthesis of an extracellular polysaccharidetermed polysaccharide intercellular adhesin [PIA] in Staphylococcusepidermidis [37, 58] and poly-N-acetylglucosamine [PNAG] in S . aureus [12, 31, 41, 43, 44], which plays an important rolein both the initial attachment and cellular proliferation processescharacteristic of staphylococcal biofilm development [27, 38,38, 40].

Much effort has been focused on understanding the regulationof ica operon expression, and it is now known that increased transcription of the ica operon can be observed under anaerobic growth conditions [13], in the presence of subinhibitory concentrationsof certain antibiotics, and in response to osmotic stress [32,52] . We have recently reported that the icaR gene encodes a transcriptional repressor with a central role in the environmental regulation of ica operon expression in S . epidermidis [10, 11].Jefferson et al . [29] demonstrated that purified IcaR proteinfrom S . aureus bound the ica operon promoter region close tothe icaA start codon . Consistent with this, deletion of theicaR gene in S . aureus is also associated with activation ofica operon expression [30; C . A . Kennedy and J . P . O'Gara, unpublished data] . A new negative regulator of ica operon expression, the teicoplanin-associated locus regulator [TcaR], has also been recently described in S . aureus [30].

The S . aureus global stress response regulator, ^B, and the S.epidermidis rsbU gene [a positive regulator of ^B] have alsobeen implicated in the regulation of biofilm development in S . aureus and S . epidermidis [32, 51] . Interestingly, inductionof biofilm by NaCl is affected in S . epidermidis rsbU and S.aureus ^B mutants [32, 51] . However, the role of ^B in the regulationof ica operon expression remains unclear given the absence ofan identifiable consensus binding site for this sigma factorupstream of the icaA or icaR start codons . In addition, Valleet al . [54] recently reported that the staphylococcal accessoryregulator, sarA, which controls the expression of over 100 genes[16], and not ^B was required for ica operon expression, PIA/PNAGsynthesis, and biofilm development in S . aureus, although ^B was found to influence the regulation of ica operon transcription. In contrast, a transposon mutation in the rsbU gene in S . epidermidiswas associated with both a reduction in PIA/PNAG levels andthe loss of biofilm-forming capacity [32].

Coagulase-negative staphylococci and S . aureus are capable of rapid phenotypic switching involving properties such as colony morphology, growth rate, antibiotic susceptibility, and biofilm-forming capacity [9, 11, 14, 25, 45, 46, 58, 59] . In terms of pathogenesis, staphylococcal phenotypic variation may contribute to dissemination, invasive disease, and sepsis . Ziebuhr et al . [59] demonstratedthat reversible inactivation of the ica operon by the insertionsequence element IS256 can result in the production of 25 to33% of phenotypic variants . More recently, we characterizedmultiple S . epidermidis isolates and demonstrated that downregulationof ica operon expression and mutation are the primary mechanismsresponsible for biofilm phenotypic variation [25] . It thereforeappears that more than one mechanism contributes to the productionof phenotypic variants in S . epidermidis . In this context, itis interesting to note the reported association between thelevels of resistance to methicillin, oxacillin, and penicillinand biofilm-forming capacity in phenotypic variants of S . epidermidis [45, 46], suggesting that the phenotypic switch associated withimpaired biofilm-forming capacity may also impact on other properties.In addition, the global regulators Agr and SarA, which influencemethicillin resistance in S . aureus [50], have also been demonstratedto regulate staphylococcal biofilm formation [2, 54, 56, 57].

In the present study we characterized the contribution of IS256 to the production of biofilm-negative variants with diminished ica operon expression . Two genetic switches were identified that involved IS256 insertions at the rsbU and sarA genes . Theimpact of IS256 insertions in both genes on biofilm, ^B regulon,and agr and sarA expression was examined . The data presentedhere provide new insights into biofilm phenotypic variationand the impact of IS256-mediated dynamic genetic events on theactivity of at least three global regulators and the regulationof staphylococcal biofilm formation.

 
Bacterial strains, media, growth conditions, and isolation of biofilm-negative phenotypic variants. The bacterial strains used in the present study are shown inTable 1 . Bacteria were routinely grown at 37°C on brainheart infusion [BHI] medium [Oxoid] supplemented with 4% ethanol,4% NaCl, or 10% NaCl . Semiquantitative determinations of biofilmformation in 96-well tissue culture plates [Nunc] were performedas described previously [11] . To screen for biofilm-negativephenotypic variants bacteria were grown on Congo red agar [CRA]plates as described previously [10, 11, 59].

 
Measurement of biofilm phase variation [switching] rates. To examine the rates of phenotypic switching, we adapted themethod of Eisenstein [17] as described previously [48] to measurethe rate at which variant cells were produced during the growthof a colony from a single biofilm-forming CFU . Black colonies from CRA were restreaked onto BHI agar and incubated overnight at 37°C . Individual colonies from these plates, which arealmost exclusively derived from an initially biofilm-positiveCFU, were resuspended and serially diluted in sterile H₂O, plated onto CRA plates, and incubated at 37°C for 24 h . Plateswere inspected by using a colony microscope to identify andcount the number of variant colonies as a proportion of thetotal number of parental, biofilm-positive, black colonies,which in turn reflects the proportion of variant and parentalcells in the original colony . Since each biofilm-positive colonyanalyzed in this way originates from a biofilm-positive bacterialCFU, the proportion of bacterial cells that have switched tothe variant phenotype in the resulting colony is a reflectionof the phenotypic switching rate . The number of generationsrequired for an individual biofilm-positive CFU to give riseto a colony is calculated by dividing the log of the total viablecount by the log of 2 [i.e., log[total viable count]/log[2]]. The data were recorded as phenotypic switch frequencies perCFU per generation.

Isolation of biofilm-positive revertants. A single colony of a biofilm-negative phenotypic variant wasgrown in BHI medium at 37°C in a tissue culture flask . After24 h the medium was replaced . This procedure was repeated untila biofilm of adhering bacteria became visible on the bottomof the tissue culture flask or at the liquid-air interface [minimumof 4 days] . After a wash with phosphate-buffered saline [PBS],the adhering bacterial cells were scratched from the bottomand streaked on CRA . After incubation at 37°C overnightand an additional 24 h at room temperature, single, black colonieswere isolated.

Genetic techniques. Genomic and plasmid DNA purification and manipulations wereperformed as described previously [10, 11] . The oligonucleotideprimers used in the present study were supplied by MWG Biotech[Germany] and are listed in Table 2.

 
The primers ICAR1 and ICAC1 were used to amplify a 4,204-bpfragment comprising the entire ica operon under the following conditions: 35 cycles of 94°C for 30 s, 47°C for 30s, and 72°C for 5 min . The primers PV1 and PV2 were usedto amplify an 871-bp product comprising the icaR gene and theica operon promoter region under the following conditions: 35cycles of 94°C for 30 s, 50°C for 30 s, and 72°Cfor 1 min.

Amplification of a 3,910-bp fragment encompassing the rsbU, rsbV, rsbW, and sigB genes was achieved by using the primersSEsigFor and SEsigRev under the following conditions: 35 cyclesof 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min . The primers rsbUFor and rsbU2 were used to amplify a 240-bp product comprising an internal portion at the 5' end of thersbU gene under the following conditions: 35 cycles of 94°Cfor 30 s, 50°C for 30 s, and 72°C for 1 min . The primersrsbUVFor and rsbUVRev were used to amplify a 1,552-bp productcomprising the rsbU and rsbV genes under the following conditions:35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°Cfor 1 min.

The sarA locus was amplified on a 1,498-bp fragment by using the primers SEsarA1 and SEsarA2 under the following conditions: 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°Cfor 2 min.

S . epidermidis strains were screened for the presence of IS256 by using a PCR assay as described previously [11].

Nucleotide sequencing was performed commercially by MWG Biotech [Germany] with the following primers: rsbUFor, rsbU2, rsbU4,rsbU5, SEsarAFor, SEsarARev, PV1, and PV2.

RNA purification and RT-PCR. RNA purification, reverse transcription-PCR [RT-PCR] and analysisof RT-PCR data was performed as described previously [10, 11], with the following oligonucleotide primer pairs: GYR1 and GYR2 [gyrB], KCA1 and KCA2 [icaA], KCR1 and KCR2 [icaR], SEasp23Forand SEasp23Rev [asp23], SEcsb9For and SEcsb9Rev [csb9], SErsbVForand SersbVRev [rsbV], SEsarAFor and SEsarARev or SEsarA3 andSEsarA4 [sarA], and SERNAIIIFor and SERNAIIIRev [RNAIII] . RTwas performed at 55°C for 30 min, followed by 14 to 24 amplificationcycles of 94°C 30 s, 50°C 30 s, and 72°C 30 sec.

Hemagglutination assays. PIA/PNAG expressed by S . epidermidis strains has been shownto be responsible for their ability to agglutinate red bloodcells [18, 31, 39] . Thus, the hemagglutination assay can beused as an indirect assay for PIA/PNAG production . Briefly, S . epidermidis RP62A cultures were grown to early stationary phase [optical density at 600 nm [OD₆₀₀] = 6.0] in BHI medium. A 1% sheep red blood [SRB] cell suspension was made by reconstituting lyophilized SRB cells [Sigma, St . Louis, Mo.] in PBS supplemented with 1% bovine serum albumin . The bacterial cultures were washed once in PBS and resuspended in PBS supplemented with an additional 2% NaCl . Then, 50 µl of the cell suspension was addedto each well in the top row of a round-bottom 96-well plate,and subsequent twofold dilutions were made in PBS supplementedwith an additional 2% NaCl . Subsequently, a 50-µl aliquotof 1% SRB was added to each well, and the plate was incubatedwithout mixing at room temperature for 2 h before visual examination.A positive result was defined as the production of diffuse redblood cells with no red blood cells pelleting at the bottomof the well.

 
Contribution of IS256 to biofilm phenotypic switching. A role for IS256 in phenotypic variation of biofilm formationin S . epidermidis was originally described by Ziebuhr et al.[58] . Insertional inactivation of the ica operon by this mobile genetic element via a unique and reversible mechanism resultsin the production of between 25 and 33% of phenotypic variantsin the reference strain RP62A [59] . More recently, we conducted an analysis of multiple S . epidermidis isolates which suggested that mutation and downregulation of ica operon expression are the primary mechanisms governing biofilm phenotypic variation[25].

To further investigate biofilm phenotypic variation in S . epidermidis strains, we measured the rate of phenotypic switching in six strains harboring IS256 [RP62A, CSF24047, SE56, 13652, 8621, and 17174] and six strains lacking IS256 [CSF41498, SE5, 14765, 1457, BC78032, and BC70837] [Table 1] . To facilitate this analysis,we adapted the method of Eisenstein [17] as described previously[48] to measure the rate at which variant cells were producedduring the growth of a colony from a biofilm-forming CFU [Materialsand Methods] . The data are expressed as the rate of switchingper CFU per generation . To assess the validity of this methodology,we confirmed that rate of phenotypic switching for S . epidermidisRP62A was ca . 5.5 x 10^–5 per cell per generation, whichwas in good agreement with previous measurements for this strain[59].

Similar to RP62A, the biofilm-positive to biofilm-negative switching frequencies in the five clinical isolates that harbor the IS256 element [CSF24047, 13652, 8621, 17174, and SE56] were between3 x 10^–5 and 5.5 x 10^–5 per CFU per generation [Table1] . In contrast, the switching frequencies in the six IS256-negativeisolates were significantly reduced compared to the IS256-positivestrains [P = 1.8 x 10^–5] [Table 1] . This low switchingfrequency in the IS256-negative strains also contrasted withthe switching frequencies reported by others in both S . epidermidis[58] and S . aureus [1], all of which were ca . 10^–5 percell per generation . These data suggest that the presence ofIS256 is generally associated with more rapid phenotypic variationof biofilm-forming capacity in S . epidermidis.

Regulation of ica operon expression and biofilm formation in a phenotypic variant of S . epidermidis RP62A. To investigate the possibility that IS256 insertions outsidethe ica operon may play a role in the more rapid productionof biofilm-negative variants with reduced ica operon transcription, we characterized the environmental regulation of ica operon expression in biofilm-negative variants of RP62A . RT-PCR was used to measure icaA and icaR transcription in variants grown in BHI broth or BHI medium supplemented with 4% NaCl, 10% NaCl, or 4% ethanol . This analysis revealed that growth of one variant, designated 33A, in the presence of 4% ethanol but not in thepresence of 4% or 10% NaCl resulted in ica operon activation[Fig. 1A] . Consistent with this, at the phenotypic level, the capacity of 33A to form biofilm was partially restored [albeit not to wild-type levels] in the presence of ethanol but notNaCl [Fig . 1B] . Interestingly, Knobloch et al . [32] also reportedthat a transposon mutation in the rsbU gene of S . epidermidis1457 resulted in a biofilm-negative phenotype, which could bereversed by growth in ethanol but not NaCl . rsbU is the firstgene of the sigB operon and encodes a phosphatase, which positivelyregulates ^B [32] . Using RT-PCR we demonstrated that, a finding consistent with the biofilm phenotypes, growth of the 1457 rsbU transposon mutant M15 only in ethanol and not NaCl was associated with activation of ica operon expression [Fig . 1C and D] . Inaddition, because PIA/PNAG expressed by S . epidermidis strainsmediates erythrocyte agglutination [18, 31, 39], the abilityof 33A to agglutinate SRB cells was tested . Consistent withthe reduced levels of ica operon transcription, hemagglutinationassays revealed a significant reduction in PIA/PNAG levels in33A compared to RP62A [data not shown] . These data suggestedthat altered ^B activity in the RP62A variant 33A may be responsiblefor diminished ica operon expression and the biofilm-negativephenotype.

 
Analysis of ^B activity in phenotypic variants. In order to assess the levels of ^B activity in the RP62A variant33A, we used RT-PCR to measure ^B-dependent gene expression.Three ^B-regulated genes were chosen for this analysis: asp23[21, 34, 47, 51], csb9 [22], and rsbV [24, 32, 49].

This analysis revealed that transcription of the asp23, csb9, and rsbV genes in the RP62A variant 33A were substantially reduced compared to the wild-type parent [Fig . 2A] . In addition, a similarlydramatic decrease in asp23, csb9, and rsbV expression was observedin the S . epidermidis rsbU transposon mutant M15 compared toits wild-type parent 1457 [Fig. 2B] . These findings stronglysuggest that ^B activity is impaired in variant 33A and thatthis change in the activity of a global regulator is responsiblefor diminished ica operon expression and a biofilm-negativephenotype.

 
PCR amplification and nucleotide sequence analysis of the sigB locus from biofilm-negative variants. Analysis of ica operon environmental regulation and ^B-dependent gene expression in the RP62A variant 33A suggested that impaired ^B activity may be responsible for the biofilm-negative phenotype.We therefore decided to characterize the sigB operon of variant 33A . The sigB operon of S . epidermidis has previously been characterizedand comprises the four genes rsbU, rsbV, rsbW, and sigB [32].Long-range PCR amplification of the sigB operon generated productsof ca . 5,200-bp for variant 33A compared to the expected sizeof 3,910 bp for RP62A [data not shown] . This observation suggestedthat a genetic rearrangement may have occurred at the sigB locusof variant 33A and was particularly interesting given that IS256 insertions in the ica operon can also generate biofilm-negative variants [59] . Restriction enzyme and nucleotide sequence analysissubsequently revealed the presence of an IS256 insertion atthe 5' end of the rsbU gene in variant 33A and thus identifiedthe genetic basis for the impaired ^B activity in this strain[Fig . 3] . Consistent with the findings of Ziebuhr et al . [59],the IS256 element, which was oriented in the opposite transcriptional orientation to the rsbU gene, was flanked by an 8-bp duplicated target sequence [Fig . 3].

 
Frequency of rsbU::IS256 insertion variant production. To determine the frequency at which rsbU::IS256 insertions occurred,we performed three independent experiments to isolate 43 biofilm-negativevariants from RP62A . PCR analysis of all 43 variants revealedthat 11 [25.5%] contained ica::IS256 insertions, whereas 5 [11.6%]contained IS256 insertions in the rsbU gene of the sigB locus.Nucleotide sequence analysis confirmed the presence of IS256insertions in the rsbU gene and identified two new IS256 insertionsites at the 5' end of rsbU in four of these variants, whichwere designated Red 1, Red 2, Red 3, and Red 4 [Fig . 3] . The fifth variant, Red 5, contained an IS256 insertion at the 3' end of the rsbU gene [Fig . 3] . In contrast to the IS256 insertionin variant 33A, IS256 was oriented in the same transcriptionalorientation to the rsbU gene in all five of these variants.Similar to 33A, we identified 8-bp duplicated IS256 target sequencesin all five of these rsbU::IS256 insertion variants; however,no consensus target sequence could be identified . These datasuggest that the rsbU gene, and in particular the 5' end ofthis gene, may represent a chromosomal hot spot for IS256 insertions.

Seventeen phenotypic variants isolated from one experiment were characterized further by using PCR to amplify the ica and sigB operons and RT-PCR to measure ica operon and asp23 expression[Fig . 4] . In this particular experiment, 3 of the 17 variantsexamined were found to contain IS256 insertions in the rsbUgene of the sigB operon [Fig. 4A] . Only one variant containedan ica::IS256 insertion [Fig . 4B], indicating that considerable variation in the rates of IS256 insertions at specific loci exists between experiments . Interestingly, RT-PCR analysis revealed that ica operon expression was substantially reduced in 14 of the 17 variants, including the three variants with rsbU::IS256 insertions [Fig . 4C] . Importantly, five variants including thethree rsbU::IS256 insertion variants were found to have reducedlevels of asp23 expression [Fig. 4C] . The molecular basis forreduced asp23 expression in the two variants that do not containIS256 insertions in the sigB operon is unknown.

 
Selection and analysis of biofilm-positive revertant strains from an rsbU::IS256 insertion variant. Ziebuhr et al . [59] reported previously that biofilm-negativeicaC::IS256 insertion variants were capable of reverting, afterrepeated passage, to a biofilm-positive phenotype after thecomplete excision of the insertion sequence element . In orderto determine whether rsbU::IS256 insertion variants could alsorevert to a biofilm-positive phenotype, we serially passagedvariant 33A in a tissue culture flask for up to 15 days . Sevenindependent flasks were used and revertants, which were isolatedfrom a weak biofilm of adhering bacteria formed after 5 to 9days, were identified as displaying a stable, black colony morphologyon CRA . Interestingly, PCR revealed that in all revertants tested,the rsbU::IS256 insertion remained intact, suggesting that asecondary or compensatory mutation was responsible for the reversionto a biofilm-positive phenotype . To further investigate thispossibility, we examined the ica operon and asp23 gene expressionin three revertants, 33A/1 to 33A/3 . Consistent with the presenceof an rsbU::IS256 allele, asp23 expression levels were substantiallylower in 33A, and all three revertants compared to wild-typeRP62A [Fig . 5] . In contrast, ica operon expression levels were predictably higher in all revertants compared to 33A but werenot comparable to the levels of ica expression in RP62A [Fig. 5] . Semiquantitative biofilm assays revealed that the revertantstrains were only weakly biofilm positive when grown in BHI broth but were strongly biofilm positive when grown in NaClor ethanol [data not shown] . Thus, the biofilm phenotype ofthese revertants was different from both 33A and wild-type RP62A.We have previously reported that the icaR gene encodes a negative regulator involved in the environmental regulation of ica operon expression [10, 11] . In order to determine whether mutationsin the ica operon regulatory regions were responsible for theincreased levels of ica operon expression in the revertant strains,we determined the nucleotide sequence of the icaR gene and icaoperon promoter region in all seven revertants . However, nosequence differences were identified in the icaR gene and ica operon promoter region of these revertants [data not shown].

 
Impact of rsbU::IS256 insertions on the expression of sarA and agr. The absence of a recognizable ^B promoter upstream of the icaoperon [32, 51] may suggest an indirect role for ^B in the regulationof ica operon transcription . In addition, Valle et al . [54]and Beenken et al . [2] recently demonstrated an essential rolefor the staphylococcal accessory regulator SarA in S . aureusbiofilm development . Valle et al . [54] also demonstrated that inactivation of sigB in S . aureus was only associated with slightlydecreased ica operon expression . Because ^B-dependent promotershave been identified upstream of the sarA gene in both S . epidermidis[19] and S . aureus [5, 15, 42], we decided to investigate theexpression of sarA in the rsbU::IS256 insertion variant 33A.This analysis revealed that sarA transcription was reduced invariant 33A compared to wild-type RP62A and that a similar patternof regulation was evident in the rsbU transposon mutant M15compared to its parental strain 1457 [Fig. 6] . Thus, these datasuggest that decreased levels of sarA transcription in phenotypicvariants harboring rsbU::IS256 insertions may also contributeto diminished levels of ica operon expression.

 
A number of studies have revealed that SarA is required formaximal expression of the two staphylococcal accessory generegulatory promoters agr P2 and agr P3 [3, 4, 6-8, 28, 35].Transcription of agrP3-driven RNAIII was also substantiallyreduced in the rsbU transposon mutant M15 and the rsbU::IS256insertion variant 33A compared to 1457 and RP62A, respectively[Fig . 6], which was consistent with the decreased levels ofsarA expression in these strains . Thus, taken together, thesedata suggest that IS256 insertions at rsbU directly affect ^B activity and accordingly sarA expression, which in turn is associatedwith reduced levels of RNAIII transcription.

Isolation and characterization of a sarA::IS256 insertion variant. To investigate the possibility that IS256 insertions at othersites may also play an important role in the production of biofilm-negativevariants with diminished ica operon expression, we used PCRto amplify the sarA locus in all 43 RP62A variants that we hadpreviously characterized [see above] . One variant, designatedRed S1, was identified in which the amplified sarA fragmentwas ca . 2,800 bp compared to the expected 1,498-bp fragmentproduced by the wild-type RP62A . Restriction enzyme and nucleotidesequence analysis subsequently confirmed the presence of anIS256 insertion within the sarA gene in variant Red S1 [Fig.7] . The IS256 element was oriented in the same transcriptionalorientation to the sarA gene and, as observed in the rsbU::IS256 insertion variants [Fig . 3], was flanked by an 8-bp duplicated target sequence [Fig . 7] . Interestingly, the RP62A sarA::IS256insertion mutant showed a bright red, smooth colony morphologyphenotype compared to the darker color of the rsbU::IS256 variantson CRA.

 
RT-PCR analysis revealed a reduction in ica operon transcription in the sarA::IS256 mutant compared to wild-type RP62A, particularlyduring early exponential growth [Fig . 8A] . Consistent with thereduced levels of ica operon transcription, hemagglutinationassays also revealed a significant reduction in PIA/PNAG levelsin the sarA::IS256 mutant compared to RP62A [data not shown].These findings suggested that SarA is a positive regulator ofica operon expression in S . epidermidis and was consistent withthe negative impact of a sarA mutation on ica operon expressionin S . aureus [54] . In contrast, decreased ica operon expression in the sarA mutant was not associated with altered regulation of icaR [Fig . 8A], suggesting that icaR expression is SarA-independent.Similarly, the IS256 insertion in sarA did not appear to resultin altered expression of sarA itself, suggesting that SarA doesnot regulate its own transcription [Fig . 8B] . Based on our earlier findings, which revealed that reduced sarA expression in rsbU::IS256 insertion variants was accompanied by decreased RNAIII expression, we also examined RNAIII transcription in the sarA::IS256 mutant.Surprisingly, this analysis only revealed no significant differencein RNAIII expression in the sarA mutant compared to wild-typeRP62A during early exponential growth [data not shown] and onlya small reduction in RNAIII expression during early stationary growth [Fig . 8C], suggesting that sarA may play a more importantrole in the regulation of agr expression in S . aureus than inS . epidermidis.

 
Interestingly, unlike the rsbU::IS256 mutants, a wild-type pattern of ica operon environmental regulation was observed in the sarA::IS256 variant grown in NaCl or ethanol [Fig . 9A], suggesting thatSarA may not be required for ica operon environmental regulation.However, biofilm assays revealed that ethanol- and NaCl-inducedica operon transcription was not accompanied by wild-type levelsof biofilm-forming capacity in the sarA mutant [Fig . 9B], suggestingthat sarA may also be required for normal PIA/PNAG synthesisor an ica-independent mechanism of biofilm formation.

 
Interestingly, PCR revealed a wild-type sarA allele [1,498-bp amplification product] in three biofilm-positive revertantsisolated from the sarA::IS256 mutant [data not shown] . Nucleotide sequencing of the sarA locus from one revertant, Red S1/R1, revealed that IS256 had been precisely excised, leaving behind a fully intact sarA gene [data not shown] . RT-PCR analysis revealed that ica operon transcription was restored to RP62A levels in revertant Red S1/R1 [Fig . 8A] . Moreover, as observed in wild-typeRP62A and unlike the sarA::IS256 insertion variant, growth ofthe revertant Red S1/R1 in NaCl or ethanol was associated withboth ica operon activation and increased biofilm formation [Fig.8].

 
Staphylococcal device-related infections involving biofilmsare not always localized to the site of the implanted biomaterialbut are sometimes characterized by dissemination of the causativeorganisms and sepsis . In this context, the contribution of phasevariation to the regulation of ica operon expression and PIAproduction might represent an important pathogenic mechanismfacilitating the release of planktonic cells from the biofilm.In the present study we investigated the molecular basis forthe production of biofilm-negative phenotypic variants withdiminished ica operon transcription . Our data reveal that inactivationof the rsbU or sarA genes by the insertion sequence elementIS256 results in diminished levels of ica operon transcriptionand is responsible for the production of ca . 11% of biofilm-negative variants in RP62A . Phenotypic variants harboring rsbU::IS256 insertions also have impaired ^B activity, as well as reducedlevels of ica, sarA, and agr expression . Importantly, IS256insertion mutations in the rsbU and sarA genes were also identifiedin biofilm-negative variants of the clinical isolate CSF24047[data not shown], suggesting that IS256 insertion mutants maybe produced by all IS256-positive strains of S . epidermidis. These findings are consistent with a recent report which revealed that IS256 may play a more significant role in staphylococcal virulence than other IS elements . These study demonstrated that not only was IS256 present in multiple copies on the genomes of disease-associated S . epidermidis strains but also that IS256 was typically associated with biofilm-forming capacity, the presence of the ica operon, and antibiotic resistance [33].

A direct role for IS256 in the ON-to-OFF switching of biofilm-forming capacity was initially characterized by Ziebuhr et al . [59], who demonstrated that reversible transposition of IS256 into hotspots within the ica operon was responsible for the production of 25 to 33% of variants . Our finding that ca . 11% of variants harbor rsbU::IS256 insertions and that IS256 can also integrateinto sarA, albeit at a lower frequency, reveals that this ISelement is responsible for the production of up to 50% of biofilm-negativevariants in S . epidermidis RP62A . The mechanism[s] responsiblefor the production of the remaining biofilm-negative variantsremains unknown . However, consistent with our previous findings[25], we were able to demonstrate that up to 80% of RP62A variantshave diminished levels of ica operon expression, indicatingthat this is the genetic basis for the biofilm-negative phenotypein the majority of variants produced by this strain.

In addition to the direct impact on ^B activity, we also obtainedevidence that the rsbU::IS256 insertion mutation indirectlyaffected the expression of two global regulators: sarA, whichis ^B dependent, and agr [RNAIII], which is SarA dependent . These data highlight the potential of IS256, through insertions at both rsbU and sarA, to alter the global regulation of transcriptionand reveal that the switch to biofilm-negative is likely tobe one of multiple phenotypic changes in rsbU and sarA IS256-generatedmutants . Interestingly, the absence of an identifiable ^B-consensus binding site upstream of the ica operon [32, 51] and the observationsthat deletion of sigB does not affect biofilm development byS . aureus [2, 54] have led to the suggestion that ^B may notdirectly regulate ica operon expression . Moreover, data whichrevealed that sarA is required for S . aureus ica operon expressionand biofilm development [54] suggested that in S . epidermidismutation of sarA or reductions in the levels of sarA transcriptionmay also contribute to repression of ica operon expression [2]. Consistent with this, analysis of the sarA::IS256 insertion mutant revealed that SarA is directly or indirectly involvedin the transcriptional regulation of ica operon expression. Comparative analysis of RP62A, 33A [rsbU::IS256], and Red S1 [sarA::IS256] revealed that ica operon transcript levels weresimilar in the rsbU and sarA mutants [data not shown] and mayfurther suggest that the impact of a ^B mutation on ica operonexpression is at least in part due to decreased expression ofsarA.

A distinctive phenotype associated with S . epidermidis rsbU mutants is the ability of ethanol but not NaCl to activate ica operon transcription . In contrast, both medium supplements activated ica operon expression in the sarA::IS256 mutant . These findingsmay suggest that ^B and SarA control ica operon expression throughseparate regulatory pathways . We have previously proposed thatica operon activation by ethanol is solely icaR dependent, whereas NaCl can activate ica expression via alterations in ^B and icaRactivity [10, 11] . The data presented in the present study suggestthat NaCl-induced ica operon activation is sarA independent.Interestingly, analysis of sarA and ^B mutants in S . aureus revealedthat PIA/PNAG levels did not reflect levels of ica transcriptionand were higher in a ^B/sarA double mutant than in a sarA singlemutant, suggesting that SarA and ^B may compete to enhance orrepress, respectively, the activity of an unknown regulatoryfactor involved in the synthesis or turnover of PIA/PNAG [54].Given that activation of ica operon expression by NaCl in theS . epidermidis sarA::IS256 variant does not result in any increasein biofilm development and that ethanol-induced ica expressionin both the sarA and rsbU mutants was not accompanied by wild-typelevels of biofilm formation, it is possible that both ^B andSarA are also involved in the posttranscriptional regulation of PIA/PNAG synthesis in S . epidermidis.

In S . aureus, sarA plays an important role in the regulation of agr transcription [4] . However, our findings only revealeda small reduction in RNAIII expression in the S . epidermidissarA::IS256 mutant . Interestingly, although expression of theS . aureus and S . epidermidis sarA genes are both driven by threeseparate promoters, one of which is ^B dependent, distinct differencespossibly reflecting functional divergence between these twoorganisms also exist [19] . For example, the sarA promoters areclustered much closer together in S . epidermidis than in S.aureus and as a result two small potential open reading frames[ORF3 and ORF4] that are present between the S . aureus sarApromoters are absent in S . epidermidis [19] . Because ORF3, together with SarA protein, may play a role in regulating agr expression in S . aureus [4], it is tempting to speculate that structuraldifferences in the sarA regulatory sequences may be reflectedin the different effects of sarA mutations on RNAIII transcriptionin S . epidermidis and S . aureus.

A recent study by Vuong et al . [55] revealed that mutation ofthe agr locus in S . epidermidis was actually associated withincreased biofilm-forming capacity and expression of the autolysinAtlE, which is involved in primary attachment [26], althoughthe levels of PIA/PNAG product were not substantially affected.Similarly the recent studies of Valle et al . [54] and Beenkenet al . [2] also demonstrated that mutation of agr did not influencebiofilm forming capacity in S . aureus . Thus, it seems unlikelythat reduced expression of RNAIII in the sarA and rsbU IS256insertion variants is an important determinant in their biofilm-negative phenotypes.

In summary, these findings provide new insights into biofilm phenotypic variation and identify two molecular mechanisms of phenotypic switching involving insertion of a transposable element into two global regulatory genes, rsbU and sarA . These genetic switches lead not only to decreased ica operon transcription and impaired biofilm-forming capacity but also reduced expression of ^B, sarA, and agr, which may in turn modulate the global regulation of transcription in this opportunistic bacterial pathogen . In addition, given that IS256 is highly active in multiresistant staphylococci and enterococci [36], our findings further highlightthe potential of transposable elements to influence the geneticflexibility and perhaps virulence of many other important gram-positivepathogens.

 
This study was funded by grants from the Research Committeeof the Royal College of Surgeons in Ireland and the Irish ResearchCouncil for Science, Engineering, and Technology to J . P . O'Gara.

We are grateful to Pfizer [Ireland] for generously supportingthe establishment of the RCSI Microbiology Laboratory at theRCSI Education and Research Centre . S . epidermidis 1457 andM15 were kindly provided by Johannes Knobloch and Dietrich Mack.Luke D . Handke and Paul D . Fey, University of Nebraska MedicalCenter, and Vance Fowler, Duke University, generously providedclinical isolates . We thank Ciara Kennedy, Sinead O'Donnell,Fidelma Fitzpatrick, and Tracey Dillane for experimental adviceand assistance throughout the study and Charles J . Dorman forcritical reading of the manuscript.

 
* Corresponding author . Mailing address: Department of Microbiology, RCSI Education and Research Centre, Beaumont Hospital, Royal College of Surgeons in Ireland, Dublin 9, Ireland . Phone: 353-1-809-3711 . Fax: 353-1-809-3709 . E-mail: jogara@rcsi.ie.

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