Molecular Microbiology First published online 27 September...

Identification and function of the RNA chaperone Hfq in theLyme disease spirochete Borrelia burgdorferimmi_7374 622..635

Meghan C. Lybecker,1 Cassandra A. Abel,1

Andrew L. Feig2 and D. Scott Samuels1,3*1Division of Biological Sciences and 3BiochemistryProgram, The University of Montana, Missoula, MT59812-4824, USA.2Department of Chemistry, Wayne State University,5101 Cass Ave, Detroit, MI 48202, USA.

Summary

Hfq is a global regulatory RNA-binding protein. Wehave identified and characterized an atypical Hfqrequired for gene regulation and infectivity in theLyme disease spirochete Borrelia burgdorferi.Sequence analyses of the putative B. burgdorferi Hfqprotein revealed only a modest level of similarity withthe Hfq from Escherichia coli, although a few keyresidues are retained and the predicted tertiary struc-ture is similar. Several lines of evidence suggest thatthe B. burgdorferi bb0268 gene encodes a functionalHfq homologue. First, the hfqBb gene (bb0268)restores the efficient translation of an rpoS::lacZfusion in an E. coli hfq null mutant. Second, the Hfqfrom B. burgdorferi binds to the small RNA DsrABb

and the rpoS mRNA. Third, a B. burgdorferi hfqnull mutant was generated and has a pleiotropicphenotype that includes increased cell lengthand decreased growth rate, as found in hfq mutantsin other bacteria. The hfqBb mutant phenotype iscomplemented in trans with the hfq gene from eitherB. burgdorferi or, surprisingly, E. coli. This is the firstexample of a heterologous bacterial gene comple-menting a B. burgdorferi mutant. The alternativesigma factor RpoS and the outer membrane lipopro-tein OspC, which are induced by increased tempera-ture and required for mammalian infection, are notupregulated in the hfq mutant. Consequently, the hfqmutant is not infectious by needle inoculation in themurine model. These data suggest that Hfq plays akey role in the regulation of pathogenicity factors in

B. burgdorferi and we hypothesize that the spirochetehas a complex Hfq-dependent sRNA network.

Introduction

Borrelia burgdorferi is the causative agent of Lymedisease (Burgdorfer et al., 1982; Benach et al., 1983;Steere et al., 1983; Radolf et al., 2010). The spirochetecycles between a tick vector and a vertebrate host (Laneet al., 1991; Spielman, 1994; Piesman and Schwan,2010), altering its gene expression to transition betweenand survive within these two vastly different environments(Singh and Girschick, 2004; Samuels and Radolf, 2009;Skare et al., 2010). The increase in temperature associ-ated with transmission from the tick to the mammal is onesignal known to increase synthesis of outer surface lipo-protein C (OspC) and other virulence factors (Schwanet al., 1995; Stevenson et al., 1995; Fingerle et al., 2000;Yang et al., 2000; Revel et al., 2002; Alverson et al., 2003;Ojaimi et al., 2003). OspC synthesis depends on the alter-native sigma factor RpoS (sS or s38), which has emergedas a global regulator of the enzootic cycle (Hübner et al.,2001; Caimano et al., 2004; 2007; Burtnick et al., 2007)and is required for infection in the murine model of Lymedisease (Caimano et al., 2004; Blevins et al., 2009). Theresponse regulator Rrp2 and the alternative sigma factorRpoN regulate the transcription of rpoS in response to anunknown environmental signal(s) (Hübner et al., 2001;Yang et al., 2003), while post-transcriptional temperature-dependent RpoS synthesis is regulated by the small RNA(sRNA) DsrABb (Lybecker and Samuels, 2007), the onlysRNA identified to date in B. burgdorferi.

Recently, sRNAs have emerged as major regulators inthe expression of many bacterial genes, including thoseinvolved in virulence and the stress response (Lease andBelfort, 2000; Hengge-Aronis, 2002; Repoila et al., 2003;Gottesman, 2004; Storz et al., 2004; Majdalani et al.,2005; Narberhaus et al., 2006; Serganov and Patel, 2007;Fröhlich and Vogel, 2009; Klinkert and Narberhaus,2009). Many sRNAs post-transcriptionally regulate geneexpression by base pairing with target trans-encodedmRNAs, affecting either their translation or stability(Repoila et al., 2003; Gottesman, 2004; Majdalani et al.,2005). In Escherichia coli, the sRNA DsrA base pairswith the rpoS mRNA upstream region, releasing the

Accepted 23 August, 2010. *For correspondence. E-mail [email protected]; Tel. (+1) 406 243 6145; Fax (+1) 406 2434184.

Molecular Microbiology (2010) 78(3), 622–635 � doi:10.1111/j.1365-2958.2010.07374.xFirst published online 27 September 2010

© 2010 Blackwell Publishing Ltd

Shine–Dalgarno sequence and start site from a stem-loopthat inhibits translation. In B. burgdorferi, DsrABb and theupstream region of the rpoS transcript have extensivecomplementarity, suggesting a similar mechanism oftranslational regulation (Lybecker and Samuels, 2007).Most trans-acting antisense sRNAs require the RNAchaperone Hfq (Massé et al., 2003a; Gottesman, 2004;Storz et al., 2004; Valentin-Hansen et al., 2004; Majdalaniet al., 2005; Romby et al., 2006; Brennan and Link, 2007;Waters and Storz, 2009). Hfq is a highly conserved RNA-binding protein that was first identified in E. coli as a hostfactor required for Qb bacteriophage replication (Franzede Fernandez et al., 1968). Hfq is the bacterial homo-logue of the eukaryotic and archaeal Sm and LSm pro-teins (Arluison et al., 2002; Møller et al., 2002;Schumacher et al., 2002; Sun et al., 2002; Zhang et al.,2002; Sauter et al., 2003). Sm and LSm proteins areinvolved in various aspects of RNA metabolism, includingprocessing of nuclear RNA, RNA localization and mRNAdecay. The Sm and LSm proteins are characterized bytwo highly conserved motifs, Sm1 and Sm2, and formcyclic ring-shaped oligomers (Achsel et al., 1999; 2001;Kambach et al., 1999). The Hfq protein is an oligomerictorus, like Sm and LSm (Törö et al., 2001; 2002).Whereas Sm and LSm proteins form heptameric struc-tures that are homomeric in archaea and heteromeric ineukaryotes, most Hfq homologues are found as homo-hexamers (reviewed in Brennan and Link, 2007).

Hfq hexamers have two independent RNA-binding sur-faces that have different specificities. The proximalsurface was defined based on the Staphylococcus aureuscrystal structure and includes the Sm2 motif (Schumacheret al., 2002). RNAs that bind this surface typically haveAU-rich single-stranded sequences either just 3′ or just 5′of a stem-loop (Brescia et al., 2003; Moll et al., 2003a;Geissmann and Touati, 2004; Vecerek et al., 2005).These RNAs bind by circumnavigating the proximal faceof the central cavity. The distal face of Hfq, on the oppositeside of the torus, also binds RNAs (Mikulecky et al.,2004). This surface, which has been characterized in arecent X-ray crystal structure, is typically associated withpoly(A) and A-rich structures (Link et al., 2009). Thecrystal structure predicts that the distal surface has speci-ficity for purine-rich repeats with the sequence (ARN)2–5.However, several other secondary structural regions havebeen identified as Hfq binding sites, including hairpinmotifs, a loop region connecting two stems in apseudoknot structure, and the T- and D-stems of tRNA(Antal et al., 2005; Lee and Feig, 2008).

Hfq functions as an RNA chaperone by facilitating theinteraction of an sRNA with its target mRNA via colocal-izing the RNAs and/or altering the structure of the RNAs(Sledjeski et al., 2001; Massé and Gottesman, 2002;Møller et al., 2002; Zhang et al., 2002; Kawamoto et al.,

2006; Vecerek et al., 2007). Hfq protects sRNAs fromdegradation likely by blocking the RNase E binding site onthe sRNA (Massé et al., 2003b; Moll et al., 2003b). Hfqalso functions in mRNA turnover by stabilizing an mRNA,stimulating turnover of the mRNA by promoting polyade-nylation, or facilitating base pairing between an sRNA andits target RNA resulting in cleavage of both RNAs byRNase E (Tsui et al., 1997; Vytvytska et al., 2000; Foli-chon et al., 2003).

An E. coli hfq null mutant has a pleiotropic phenotypethat includes decreased growth rate, increased cell lengthand increased sensitivity to stress conditions (Tsui et al.,1994; Muffler et al., 1997). Hfq is required for post-transcriptional regulation of rpoS and therefore the syn-theses of more than 50 proteins are affected by hfqmutations (Muffler et al., 1996; Brown and Elliott, 1997).Moreover, Hfq plays an important role in several patho-genic bacteria: hfq mutations either attenuate or abolishvirulence (reviewed in Chao and Vogel, 2010). Hfq islargely conserved in bacteria (Sun et al., 2002); however,B. burgdorferi lacks an annotated hfq gene in its genome(Fraser et al., 1997) and BLAST searches using sequencesof conserved hfq genes do not reveal a homologue. Wenow identify and characterize an unusual Hfq in B. burg-dorferi that regulates RpoS and is required for murineinfection by needle inoculation.

Results

Identification of Hfq in B. burgdorferi

The RNA chaperone Hfq is typically required for RNA–RNA interactions between small regulatory RNAs andtheir cognate target mRNAs. After identifying and charac-terizing the first sRNA, DsrABb, in B. burgdorferi, wehypothesized that the spirochete has an Hfq despite thelack of an annotated, or readily identifiable, homologue inits genome. This was supported by an MFOLD (Zuker,2003) secondary structure prediction of DsrABb thatrevealed a putative Hfq binding site, an AU stretch ofresidues situated between two stable stem-loops (Fig. 1).BLAST searches of the B. burgdorferi genome were per-formed using as queries the Hfq sequences from E. coliand S. aureus, as well as just the highly conservedN-terminal 68 amino acids of E. coli Hfq. No homologoussequences were found in B. burgdorferi (data not shown).We next searched the genome for small open readingframes (ORFs) that had the potential to form Sm1 andSm2 motifs, and we identified a conserved hypotheticalORF encoded by the bb0268 gene, which was predictedto have 33% similarity and 12% identity with E. coli Hfq. ACLUSTALW alignment revealed that several importantamino acids are conserved and a Phyre prediction sug-gested that this ORF could fold into an Hfq-like structure

B. burgdorferi Hfq 623

© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 78, 622–635

(Fig. 2). The residues comprising the nucleotide-bindingpocket of Hfq, in the S. aureus Hfq–RNA complex (Schu-macher et al., 2002), are conserved or similar in thepredicted BB0268 protein. Other Hfq motifs (Sm1 NGand Sm2 F/YKHA) are partially conserved in BB0268.

However, the BB0268 protein (159 amino acids) is con-siderably larger than most Hfq proteins (typically 77–110amino acids). The N-terminal 68 amino acids of knownHfq homologues are highly conserved and include the Smmotifs, while the C-terminal portions of the proteins vary inlength and sequence (Sauter et al., 2003). The CLUSTALW

alignment demonstrated that the BB0268 sequence ismost similar to the N-terminal domain of the canonical E.coli Hfq protein, but BB0268 has a longer C-terminal tailwith a divergent sequence (Fig. 2A).

B. burgdorferi bb0268 complements an E. colihfq mutant

In E. coli, Hfq is required for DsrA to stimulate translationof rpoS in response to a decrease in temperature andentrance into stationary phase (Sledjeski et al., 2001).Evidence suggests that Hfq colocalizes rpoS and DsrA,presenting the two RNAs in favourable positions forcomplex formation (Brescia et al., 2003; Lease andWoodson, 2004). To determine if BB0268 functions as anRNA chaperone, similar to Hfq in E. coli, we assayed itsability to trans-complement an hfq null mutant of E. coli

A-G-U-A-A

AAAAUUUAAUUUA

UUUUAAAUUAAAUU- -U

GUAAAU

CAUUUAA-A-A

U

U A

G

5' 3'

Fig. 1. Predicted secondary structure of DsrABb. The arrows flankthe rpoS complementary region (34 nucleotides). The sequencepredicted to be the Hfq binding site is in bold. The open circlerepresents a loop of 32 nucleotides.

Fig. 2. Hfq structural homology.A. CLUSTALW alignment of the BB0268conserved hypothetical protein with the E. coli(Ec) Hfq protein. Amino acids denoted byasterisks (*) are identical, colons (:) stronglysimilar and dots (.) weakly similar. Thedashed boxes indicate the Sm1 and Sm2motifs and the secondary structure of the E.coli Hfq is indicated above the sequence. Twoof the signature motifs of Hfq, the Sm1 NGand the Sm2 F(Y)KHA, are underlined. Theresidues that are important in RNA binding bythe S. aureus Hfq are shown in grey boxes.This figure panel is modified from Nielsenet al. (2007).B. Modelling of Hfq structures. The structureof HfqBb (grey) was predicted using Phyre andsuperimposed onto the experimentallydetermined S. aureus Hfq (blue) and E. coliHfq (cyan) structures using Chimera. Hfqhomologues from many organisms have avariable C-terminal extension that has notbeen structurally characterized; therefore, wehave no information on the folded structure ofthe C-terminal region of HfqBb and it is notshown.

B

A

Hfq Ec MAKGQSLQDPFLNALRRERVPVSIYLVNGIKLQG-QIESFDQFVILLKN-TVSQMVYKHA BB0268 MFISRELKYILLTSVSALFISAVLGLFCGLSFFTILVRSLLQFVFFFVIGLLIEYIFKKY

*:

Hfq Ec ISTVVPSRPVSHHSNNAGGGTSSN--------YHHGSSAQNTSAQQDSE----------- BB0268 LSNLFSTELSGNMENKPQDDKKSDKDFKENLDFQNKNSLYENSQNISSSGDFIEEVKKYK

Hfq Ec -ETE----------------------------------- BB0268 FETEDVSRGSDKNKKMSFIEDNDPKVVADAIKTLMSKKE

* .:.*: :*.:: :.. : *. *:.: :.*: ***::: : : ::

:*.:..:. .: .*:. ....*: ::: .* :.* : .*.

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α1 β1 β2 β3 β4

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624 M. C. Lybecker, C. A. Abel, A. L. Feig and D. S. Samuels �


(Sledjeski et al., 2001). The bb0268 gene was fused to anarabinose-inducible promoter in the vector pBAD24. Theempty pBAD24 vector and conserved hypothetical ORFbb0260, which shares sequence similarity with bb0268,were used as negative controls; E. coli hfq was used as apositive control. Hfq is needed for growth phase-relatedregulation of rpoS::lacZ at 30°C in E. coli. The efficienttranslation of an rpoS::lacZ fusion, in response tochanges in cell density, was measured with a b-galactosidase assay. bb0268 partially restored the effi-cient translation of rpoS::lacZ at 30°C (Fig. 3), suggestingthat it has an Hfq-like RNA chaperone activity in E. coli. Incontrast, neither bb0260 nor the empty vector comple-mented the hfq null mutant in E. coli. Five independentexperiments with bb0268 yielded similar results. There-fore, we now refer to BB0268 as HfqBb.

Recombinant HfqBb binds rpoS mRNA and DsrA

The Hfq and Sm/Lsm family of proteins form homohex-americ and heteroheptameric oligomers (Tsui et al., 1994;Sun et al., 2002; Valentin-Hansen et al., 2004). We puri-fied recombinant HfqBb using an N-terminal His-tag. Thepredicted molecular mass of HfqBb is ~20 kDa (with theHis-tag) and gel filtration chromatography indicated that ithas a molecular mass of ~34 kDa in solution (Fig. 4),suggesting that HfqBb forms a dimer or a monomer, ratherthan the characteristic hexamer, under these in vitroconditions. In contrast, E. coli Hfq was primarily a

b-g

ala

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Mille

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pBADHfq pBAD24 pBAD268 pBAD260

-arabinose+arabinose

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100

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Plasmid

Fig. 3. BB0268 has Hfq-like activity in E. coli. hfq-1 mutant strainscontaining an rpoS::lacZ translational fusion and carrying theindicated plasmids were assayed for total b-galactosidase activity.pBADHfq expresses E. coli Hfq under control of the araBADpromoter. pBAD24 is the empty araBAD vector. pBAD268 andpBAD260 have the B. burgdorferi bb0268 and bb0260 ORFs underthe control of the araBAD promoter. Cultures were grown at 30°Ceither with (black bars) or without (grey bars) 150 mM arabinoseand samples were taken at various times during logarithmic growth.Total b-galactosidase activity was determined as described byMiller (1972). Three independent experiments with BB0260 and fiveindependent experiments with BB0268 were performed and yieldedsimilar results.

Fig. 4. HfqBb is not a stable hexamer insolution.A. Standard curve of the Sigma gel filtrationmolecular weight markers resolved on aSuperdex 75 HR 10/30 column. Regressionanalysis yielded the equation:y = 869.58 ¥ e(-2.1536x), which was used tocalculate molecular mass.B and C. Recombinant HfqBb (B) and E. coliHfq (C) were resolved on a Superdex 75 HR10/30 column and fractions analysed using aHisProbe-HRP.

Ve/Vo

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hexamer as analysed by gel filtration chromatographyunder the same conditions, although monomer was alsoobserved in this experiment (Fig. 4C).

Electrophoretic mobility shift assays (EMSAs) were per-formed using recombinant HfqBb protein and in vitro tran-

scribed rpoS and DsrABb RNAs (Fig. 5). The RNAs wereheated to 65°C for 10 min and slow-cooled to roomtemperature. The RNAs were then incubated with increas-ing amounts of HfqBb protein. The HfqBb protein boundboth rpoS and DsrABb as seen by the decreased migrationof the RNAs in the EMSA (Fig. 5).

Construction and complementation of the hfqBb mutant

To determine the function of HfqBb in B. burgdorferi, wedeleted the hfqBb gene in a low-passage infectious cloneof strain 297 (Fig. 6A). Two different plasmids were con-structed to complement the hfqBb mutation. The hfqBb

(bb0268) and the E. coli hfq ORFs were fused to theinducible flac promoter and inserted into the shuttle vectorpKFSS1-lacI, which carries the lacI gene fused to theconstitutive flgB promoter (Fig. 6B). The two complement-ing plasmids and the parental shuttle vector were trans-formed into the hfqBb mutant strain. RT-PCR analysisdemonstrated that the wild-type and hfqBb-complementedstrains, but not the hfqBb mutant strain, expressed hfqBb

transcript (Fig. 6C).

DsrA:Hfq

DsrA

[Hfq] (µM) 0 1.5 3 6 8 10 12 14

A

B rpoS:Hfq

rpoS

[Hfq] (µM) 0 1.5 3 6 8 10 12 14

Fig. 5. The HfqBb protein binds RNA. Gel mobility shift analyses ofHfqBb binding to DsrABb (A) and rpoS (B). The RNAs were labelledwith [a-32P]-UTP during in vitro transcription, gel-purified, andincubated with increasing concentrations of purified recombinantHfqBb. The binding reactions were fractionated on a native Novex®

Pre-Cast 6% DNA retardation gel.

c p9

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bb0269bb0267

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Fig. 6. Mutation and complementation of hfqBb.A. Strategy for disrupting the hfqBb gene (bb0268). The hfqBb gene was replaced with the kanamycin-resistant cassette (flgBp-aphI ).Transformation into wild-type (WT) B. burgdorferi and homologous recombination generated an hfqBb mutant strain (hfq-).B. Plasmid for trans-complementing the hfqBb mutant strain. The B. burgdorferi or E. coli hfq genes were fused to the inducible promoter flacpand inserted into the modified shuttle vector pKFSS1-lacI, which carries the lacI repressor gene fused to the constitutive flgB promoter in themultiple cloning site. The plasmid also carries both a B. burgdorferi replication origin (cp9 rep) from the 9 kb circular plasmid and an E. colireplication origin (ColE1) from pCR®-XL-TOPO.C. RT-PCR of hfq mRNA in the wild type (WT), the hfqBb mutant (hfq -) and the hfqBb mutant complemented with hfqBb (hfq- pKFSS1-lacIflacphfqBb). The RT-PCR (+RT), the PCR on samples that were not treated with reverse transcriptase (-RT) and the PCR without template (-)were fractionated on a 2% agarose gel and stained with ethidium bromide.



The hfqBb mutant has a decreased growth rate andincreased cell length

We assayed growth rate using a wild-type low-passageinfectious clone of 297, the hfqBb mutant and the twocomplemented strains. B. burgdorferi cultures were grownat 23°C to a low cell density (1–3 ¥ 107 cells ml-1) andthen inoculated into a culture at 37°C at a cell density of1 ¥ 103 cells ml-1. Cell densities were determined dailyuntil cultures reached stationary phase. The hfqBb mutantgrew slower than the wild type; both the B. burgdorferi andE. coli hfq genes complemented the growth phenotype ofthe hfqBb mutant (Fig. 7). The hfqBb mutant strain also wassignificantly longer than wild type. The cell length pheno-type was fully complemented by the hfqBb gene expressedin trans and, somewhat unexpectedly, partially comple-mented by the E. coli hfq gene (Fig. 8).

RpoS and OspC are regulated by HfqBb

DsrABb post-transcriptionally regulates RpoS levels inresponse to increased temperature at low cell density(Lybecker and Samuels, 2007). This sRNA has extensivecomplementarity to the upstream region of the rpoSmRNA and our data suggest that DsrA stimulates efficienttranslation of the rpoS mRNA through base pairing withthe upstream region of the mRNA. Hfq in other bacteria is

Time (days)

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105

106

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108

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WThfq-hfq- pKFSS1-lacI/flacphfqBbhfq- pKFSS1-lacI/flacphfqEc

Fig. 7. Growth phenotype of the hfqBb mutant. Growth curve of thewild type (WT), hfqBb mutant (hfq -), and hfqBb mutanttrans-complemented with either hfq from B. burgdorferi (hfq-

pKFSS1-lacI/flacphfqBb) or hfq from E. coli (hfq- pKFSS1-lacI/flacphfqEc) following a temperature shift to 37°C. The means ofthe cell densities from three independent experiments are graphedwith error bars representing the standard error of the means.

Fig. 8. Cell morphology phenotype of thehfqBb mutant. Cultures were grown at 37°C tomid-log phase and the cells were visualizedusing Nomarski microscopy and analysedusing ImageJ.A. Microscopic images of the wild-type (WT),hfqBb mutant (hfq -) and complementing strains(pKFSS1-lacI/flacphfqBb or Ec).B. ImageJ was used to measure the length of50 spirochetes per strain and the means ofthe lengths are graphed with error barsrepresenting the standard error of the means.Differences between wild-type and hfqBb

mutant strains and between hfqBb mutant andpKFSS1-lacI/flacphfqBb-complemented strainsare statistically significant (P < 0.01) by both aone-way ANOVA with a Tukey’s post hoc testand a Kruskal–Wallis test; differencesbetween hfqBb mutant andpKFSS1-lacI/flacphfqEc-complemented strains(P < 0.01) and between wild-type andpKFSS1-lacI/flacphfqEc-complemented strains(P < 0.001) are significant by both a two-tailedunpaired t-test and a Mann–Whitney U-test.

hfq - pKFSS1-lacI/flacphfqEc

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required for regulating trans-encoded sRNAs and oftenpost-transcriptionally regulates RpoS levels, so weexpected that the hfqBb mutant strain would show reducedlevels of RpoS and OspC proteins in response to atemperature shift at a low cell density, as seen in thedsrABb mutant strains. B. burgdorferi low-passage wild-type, hfqBb mutant and complemented strains weretemperature-shifted to 37°C and grown to low andhigh cell densities (1–3 ¥ 107 and 1 ¥ 108 cells ml-1

respectively). Western blot analyses of whole-cell lysatesdemonstrated that the wild-type and complementedstrains displayed an increase in RpoS and OspC levelsafter a temperature shift, while the hfqBb mutant strainshowed reduced levels of RpoS and OspC (Fig. 9A). Sur-prisingly, the E. coli hfq also restored the temperature-dependent increase in RpoS and OspC. The inducible

promoter fusions are leaky in these trans-acting configu-rations (Fig. 6C and data not shown), so IPTG was notrequired for transcription of the complementing hfq genes.Adding high levels of IPTG caused decreases in RpoSand OspC levels, presumably due to the overabundanceof Hfq inhibiting translation (data not shown).

Transcription of rpoS requires RpoN and Rrp2 (Hübneret al., 2001; Yang et al., 2003). To further understand themechanism by which HfqBb regulates RpoS, the steady-state levels of rpoS, rpoN and rrp2 mRNA were analysedby Northern blot analyses (Fig. 9B). The constitutivelyexpressed flaA transcript was used as a gel loadingcontrol. The amounts of the rpoN and rrp2 transcriptswere similar between the wild-type, mutant and comple-mented strains demonstrating that Hfq does not regulatethe levels of rpoN or rrp2 mRNA. However, the levels ofrpoS transcript were 1.5-fold higher in the hfqBb mutantstrain compared with the wild-type and complementedstrains; this pattern of regulation was also observed in thedsrABb mutant strain (Lybecker and Samuels, 2007).These data, taken together, suggest that HfqBb regulatesRpoS synthesis post-transcriptionally and does not stabi-lize the rpoS mRNA.

As previously seen with the dsrABb mutant (Lybecker andSamuels, 2007), serial passage of the hfqBb mutant resultsin a reversal of the phenotype. B. burgdorferi cultures wereserially passaged at 23°C and then temperature-shifted to37°C and grown to a low cell density. Western blot analysesdemonstrated that both RpoS and OspC syntheses wereincreased, rather than decreased, in the mutant comparedwith the wild type after as few as six passages (data notshown). The mechanism by which the phenotype switchesis not yet understood.

HfqBb is required for mouse infection byneedle inoculation

Several bacterial species require Hfq for virulence (Chaoand Vogel, 2010). We hypothesized that the hfqBb mutantstrain would not be infectious in mice due to the absenceof RpoS and OspC. We assayed the infectivity of the hfqBb

mutant in the mouse model of Lyme disease by needleinoculation (Barthold et al., 2010). B. burgdorferi cultureswere temperature-shifted to 37°C and grown to a low celldensity (1–3 ¥ 107 cells ml-1); C3H-HeJ female mice wereneedle inoculated with 5 ¥ 103 cells intraperitoneally.Tissue samples were cultured and assayed for the pres-ence of B. burgdorferi by dark-field microscopy; culturesthat were negative by this method were also screened byPCR (Wang et al., 2010). The hfqBb mutant was unable toinfect mice and complementation with hfq from B. burg-dorferi, but not E. coli, restored infectivity (Table 1). Thus,hfqBb is essential for B. burgdorferi infection of mice byneedle inoculation.

kDa207

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Temp (˚C) 23 37 37 23 37 37 23 37 37 23 37 37

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B

Fig. 9. HfqBb regulates RpoS levels post-transcriptionally inresponse to a temperature shift.A. Immunoblot analyses of whole-cell lysates of wild-type, hfqBb

mutant, and trans-complemented strains at 23°C and after atemperature shift to 37°C at low (L) and high (H) cell densities(1–3 ¥ 107 and 1 ¥ 108 cells ml-1 respectively). The top panel is aCoomassie Brilliant Blue-stained SDS-PAGE gel and the bottompanels are immunoblots probed with either anti-RpoS or anti-OspCantibodies. Three independent experiments were performed andrepresentative data are shown.B. Northern blot analyses of total RNA (15 mg) fractionated on a 6%polyacrylamide 7 M urea gel, blotted to a nylon membrane, andhybridized with an rpoS, rpoN, rrp2 or flaA single-stranded RNAprobe. Two independent experiments were performed andrepresentative data are shown; the signal was quantified on aFujiFilm LAS-3000.



The high-passage hfqBb mutant retained the plasmidsessential for infectivity and was used to needle inoculatemice. RpoS and OspC are highly expressed in the high-passage hfqBb mutant, so we expected it to be infectious,but the strain did not infect the mice (Table 1). These dataand the finding that the hfqBb mutant complemented withhfq from E. coli was avirulent, even though it expressedwild-type levels of RpoS, suggest HfqBb may play addi-tional roles in mammalian infection besides regulation ofRpoS.

Discussion

The RNA chaperone Hfq has emerged as an importantglobal post-transcriptional regulator in mRNA translationalcontrol by sRNAs (Valentin-Hansen et al., 2004). In thisstudy, we identified and characterized an unusual Hfq in B.burgdorferi. The HfqBb primary sequence does not have ahigh degree of similarity or identity to annotated Hfq homo-logues, but does retain a few key residues and was pre-dicted to have a superimposable tertiary structure (Fig. 2).A BLAST search using full-length HfqBb, or the more highlyconserved N-terminal region of HfqBb, as the query onlyidentified hypothetical ORFs within the spirochete phylum(data not shown). Hfq is characterized as a part of theSm/Lsm protein family and these proteins form ring-shaped oligomeric structures that are homohexameric inbacteria and heteroheptameric in eukaryotes. Despitethese differences, the Hfq and Sm/Lsm monomers fold intoa similar structure, consisting of an N-terminal a-helixfollowed by a curved five-stranded b-sheet (Schumacheret al., 2002; Sauter et al., 2003). Our gel filtration datasuggested that recombinant HfqBb does not form a stablehexamer in solution (Fig. 4), which is unlike most othercharacterized Hfq homologues. However, we hypothesizethat the functional quaternary structure of HfqBb in vivo is ahomohexamer, which is unstable under our experimentalconditions. Possibly, our inability to observe a hexamer invitro is due to the presence of a lysine residue rather thanthe conserved histidine at position 57 (in the E. colisequence), which is known to be involved in stabilizing thehexameric structure (Moskaleva et al., 2010). Despite thedifferences in primary and quaternary structure between

HfqBb and the canonical Hfq, our results clearly establishthat HfqBb functions as an RNA chaperone as demon-strated by hfqBb complementing an rpoS translationaldefect in an hfq null mutant of E. coli (Fig. 3). Moreover, E.coli hfq complemented the hfqBb mutant phenotype withregard to growth rate (Fig. 7), cell length (Fig. 8), and RpoSand OspC induction (Fig. 9). This is the first time, to ourknowledge, that a heterologous bacterial gene hascomplemented a B. burgdorferi mutant.

The hfqBb mutant has a phenotype that includesdecreased growth rate, increased cell length, alteredRpoS regulation and loss of infectivity. The B. burgdorferiand E. coli hfq genes complemented the in vitro mutantphenotype demonstrating that the phenotype is due to theloss of hfqBb; these phenotypes have been observed inseveral hfq mutants of other bacteria (Brennan and Link,2007; Chao and Vogel, 2010). RpoS is required for mam-malian infection by B. burgdorferi, and therefore the hfqBb

mutant was not expected to be infectious in the mousemodel. Our findings established that HfqBb is required formammalian infectivity, but that the mechanism is notsolely due to the loss of RpoS induction. A high-passagehfqBb mutant strain that produces RpoS and OspC wasstill not infectious in mice (Table 1). These data demon-strated that HfqBb is required for infectivity in mice inde-pendent of RpoS and OspC expression. We hypothesizethat there are other Hfq-dependent sRNAs in B. burgdor-feri that regulate expression of virulence factors. In E. coliand Salmonella typhimurium, Hfq has other regulatoryfunctions independent of its effects on RpoS expression(Muffler et al., 1997; Sittka et al., 2007). Hfq in the patho-genic bacteria S. typhimurium, Vibrio cholerae and Bru-cella abortus is required for full virulence, but themechanism is independent of the requirement of Hfq forRpoS expression (Robertson and Roop, 1999; Ding et al.,2004; Sittka et al., 2007).

We hypothesized that HfqBb regulates RpoS synthesisvia the sRNA DsrABb and, thus, we expected the RpoSdefect would be similar in the dsrABb mutant and hfqBb

mutant strains. Our data demonstrated that HfqBb post-transcriptionally regulates the temperature-dependentincrease in RpoS. Surprisingly, unlike the dsrABb mutantstrain, which only shows reduced levels of RpoS at a lowcell density (log-phase growth), the hfqBb mutant strainhad reduced levels of RpoS at both low and high celldensity (log and stationary growth phase respectively),suggesting that HfqBb also plays a DsrABb-independentrole in RpoS regulation.

In conclusion, we have identified and characterized anunusual Hfq protein in the Lyme disease spirochete B.burgdorferi that is essential for infectivity. Although onlyone sRNA has been identified to date in B. burgdorferi, thepleiotropic phenotype of the hfqBb mutant suggests othersRNAs exist and likely play a role in the enzootic life cycle.

Table 1. HfqBb is required for mouse infection by needle inoculation.

No. of positive mice by culture

B. burgdorferi strain Ear Joint Bladder

297 (BbAH130) WT 6/6 6/6 6/6297 hfq- 0/6 0/6 0/6297 hfq- high passage 0/3 0/3 0/3297 hfq-/flacp-hfqBb 3/3 3/3 3/3297 hfq-/flacp-hfqEc 0/3 0/3 0/3



Experimental procedures

B. burgdorferi strains and culture conditions

Borrelia burgdorferi low-passage virulent strain 297 (cloneBbAH130) (Hübner et al., 2001) and all derivatives used in thisstudy were cultivated in Barbour–Stoenner–Kelly II (BSK-II)complete medium (Barbour, 1984) at either 23°C or 37°C.Cultures were passaged to 1 ¥ 105 cells ml-1 from 23°C to37°C and grown to either 1–3 ¥ 107 cells ml-1 (low density),which corresponds to mid-log phase, or about 1 ¥ 108

cells ml-1 (high density), which corresponds to stationaryphase. For growth curves, cultures were grown at 23°C to1–3 ¥ 107 cells ml-1 and passaged to 1 ¥ 103 cells ml-1 at37°C. Cell density was determined using a Petroff Haussercounting chamber (Hausser Scientific Partnership) as previ-ously described (Samuels, 1995). All cultures were counted intriplicate and at least three independent experiments wereperformed.

Sequence analysis and structural modelling

The CLUSTALW sequence alignment was performed withNPS@: Network Protein Sequence Analysis (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.

html) (Combet et al., 2000). A computational prediction of theHfqBb fold was performed using Protein Homology/analogYRecognition Engine (Phyre) (http://www.sbg.bio.ic.ac.uk/~phyre/) (Kelley and Sternberg, 2009). The resulting PDBcoordinate file covering residues 6–71 was then visualizedand overlaid with the structures of Hfq from E. coli (PDB1HK9) (Sauter et al., 2003) and Hfq from S. aureus (PDB1KQ2) (Schumacher et al., 2002) using Chimera (http://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004).

E. coli strains and plasmid construction forhfq complementation

Escherichia coli strain DDS1631 has an hfq null mutation andan rpoS::lacZ translational fusion (Sledjeski et al., 2001). ThepBADHfq plasmid (pDDS400) carries the E. coli hfq genecloned into the EcoRI and PstI sites of the arabinose-inducible vector pBAD24 (Sledjeski et al., 2001). The bb0268and bb0260 genes were PCR-amplified using the proof-reading enzyme KOD with engineered MfeI and PstI sites atthe 5′ and 3′ ends respectively (Table 2). After poly(A) tailingwith Taq, the PCR products were cloned into pCR®2.1-TOPO.Positive transformants were screened by PCR and confirmedby DNA sequencing. The pBAD260 and pBAD268 plasmids

Table 2. Oligonucleotides used.

Name Sequence (5′-3′)

Complementation in E. coliBB268 1F+MfeI ACAATTGACCATGTTTATAAGCAGGGAATTGBB268 480R+PstI ACTGCAGTTATTCCTTCTTGCTCATTAAAGBB260 1F+MfeI ACAATTGACCATGAAAGGGTTTTTAGCGBB260 1014R+PstI ACTGCAGTTAAACATATTGATCTTTTTC

Complementation, expression, and RT-PCRBB268 1F+NdeI CATATGTTTATAAGCAGGGAATTGBB268 480R+AatII GACGTCTTATTCCTTCTTGCTCATTAAAG

RT-PCRBB268 249R+AatII GACGTCTTAACTCTTCTTATCATCTTG

Complementation in B. burgdorferiEchfq 1F+NdeI CATATGGCTAAGGGGCAATCTTTACEchfq 310R+AatII GACGTCTTATTCGGTTTCTTCGCTGTC

Mutant constructionBB268 U1048F TTTCTTATGTTACGGATGGGCBB268 U4R+Aat+Age ACCGGTCATGACGTCTTATTCCACACCAAAAAATCBB268 474F+AatII GACGTCGGAATAAATATGGATGACAGGGCBB268 D1578+AgeI ACCGGTTACTATATCTCCAGTAGCAGGTCC

Template for in vitro transcriptiondsrA L1F+T7 TAATACGACTCACTATAGGGAAAGCTAAATTAGAAGAGTTTATTGdsrA L275/352R CATAAAAACTTTTTTTTGAATAGrpoS U449F+T7 TAATACGACTCACTATAGGGTCACAAAATCTAAAATTTAAAAATCrpoS 128R GTTTTTTGCTTTTGCATTGC

Northern blot probesrpoS 705R+T7 TAATACGACTCACTATAGGCGGTGCTTTTTTTGGGACTATTGrpoS 443F GATTCAACCTATCTCCTGCTCAGrrp2 185F GAGAAAAATTGCTCAAAATArrp2 394R+T7 TAATACGACTCACTATAGGCTTTTGAGTTTTTTCAAGAAGrpoN 285F GCAATTAAGAATTCAAAGAArpoN 572R+T7 TAATACGACTCACTATAGGCTTTTGAGTTTTTTCAAGAAGflaA 64F GCTCAAGAGACTGATGGATTAGCflaA 284R+T7 TAATACGACTCACTATAGGCGCAGAAGGAGTAAGTAAAACGCTC



were generated by directionally cloning the MfeI–PstI excisedbb0268 and bb0260 fragments into the EcoRI and PstI sitesof pBAD24.

E. coli Hfq complementation

b-Galactosidase units were assayed using the Zhang andBremer (1995) modification of the Miller (1972) assay. Briefly,cells were grown with shaking at 30°C in lysogeny brothmedium supplied with kanamycin and carbomycin in thepresence or absence of 150 mM arabinose. The cultures wereinoculated 1:100 from overnight cultures and samples weretaken at an OD600 ranging from 0.4 to 1.0 (approximately logphase). The OD600 of each culture was measured and 30 mlaliquots were removed and mixed with 70 ml of permea-bilization solution [100 mM Na2HPO4, 20 mM KCl, 2 mMMgSO4, 0.8 mg ml-1 CTAB (hexadecyltrimethylammonium),0.4 mg ml-1 sodium deoxycholate and 5.4 ml ml-1 2-mercaptoethanol] and stored at room temperature. Thesamples were incubated at 30°C for 30 min. The substratesolution [60 mM Na2HPO4, 40 mM NaH2PO4, 1 mg ml-1

o-nitrophenyl-b-D-galactoside (ONPG) and 2.7 ml ml-1

2-mercaptoethanol] was also incubated at 30°C for 30 minand 600 ml of substrate solution was added to each sample.After sufficient colour developed, 700 ml of stop solution (1 MNa2CO3) was added to each sample and the time wasrecorded. The samples were centrifuged at 14 000 r.p.m. inan Eppendorf 5417R for 5 min to remove cell particulates.The OD420 (between 0.05 and 1.0) was measured for eachsample and the total b-galactosidase units were calculatedfrom the formula:

1000 OD OD of culture volume of culturereact

420 600× ( ) ÷ ( ) × ( ) ×[iion time .( )]

The volumes of permeabilization solution and cell culturevaried between experiments and were appropriately changedin the calculation of total b-galactosidase units.

Cloning and protein purification of Hfq

The hfqBb gene was amplified by PCR with the proof-readingenzyme KOD, poly(A) tailed and cloned into pCR®2.1-TOPO(Table 2). The hfqBb gene was directionally cloned into aderivative of pET28b with an N-terminal, tobacco etch virusprotease-cleavable His6 tag (Corbett et al., 2004) using anengineered NdeI site at the 5′ end of the gene and thepCR®2.1-TOPO vector BamHI site. The resulting plasmidpET28b-TEV::Hfq was sequenced and transformed into E.coli Rosetta (DE3) cells (Novagen). Cell cultures were grownat 37°C to an OD600 of 0.4–0.6, induced with 1 mM IPTG andgrown overnight at 11°C and again overnight at 18°C. Cellswere lysed using Bugbuster (Novagen), filtered through a0.45 mm filter and loaded directly onto 1 ml Ni2+ column(HisTrap HP, Amersham Biosciences). The resin was washedwith 10 column volumes of 20 mM NaH2PO4, 500 mM NaCland 5 mM imidazole, pH 7.4, five column volumes of 1 MNH4Cl, and five column volumes of 1 M urea, and finallyeluted with a 10 ml linear gradient of 10–500 mM imidazole,pH 7.4, in 20 mM NaH2PO4 and 500 mM NaCl. The fractionscontaining Hfq were dialysed into a storage buffer (20 mMNaH2PO4 and 125 mM NaCl, pH 7.4) and concentrated in

Amicon Ultra-4 spin columns. The concentration of the puri-fied recombinant protein was determined with the BCAprotein assay (Pierce). The E. coli Hfq protein was purified aspreviously described (Mikulecky et al., 2004).

Gel filtration

Recombinant purified HfqBb and E. coli Hfq were dialysedovernight in 50 mM Tris-HCl, pH 7.5 and 100 mM KCl. Sigmagel filtration molecular weight markers were run on a Super-dex 75 HR 10/30 column and a standard curve was plotted.The Hfq proteins were run on the column and the molecularmass was calculated using the standard curve.

Template preparation for in vitro transcription

Borrelia burgdorferi strain 297 total DNA was used as atemplate in PCR with the T7 bacteriophage promoter addedto the 5′ end of either the forward primer to generate senseRNAs for the EMSA or the reverse primer to generate anti-sense RNAs for probes in Northern blots (Table 2). The PCRproducts were visualized on an agarose gel stained withethidium bromide. If a single product was visualized, the PCRproduct was precipitated with ammonium acetate and resus-pended in DEPC-treated water. If more than one product wasvisible, the appropriate band was purified with the Qiagen gelpurification kit following the manufacturer’s protocol andresuspended in DEPC-treated water.

In vitro synthesized RNA

DsrABb and rpoS RNAs for EMSAs were synthesized andradioactively labelled by incorporation of [a-32P]-UTP duringin vitro transcription using the T7-Megascript In Vitro Tran-scription kit (Ambion) according to the protocol suggested bythe manufacturer. PCR products (described above) wereused as templates for the in vitro transcription reactions.Reaction time was increased to overnight at 37°C to increasethe yield of short products. Following in vitro transcription, thefull-length RNA transcripts were resolved on a 6%polyacrylamide-7 M urea gel and eluted from gel slices byincubating in elution buffer (20 mM Tris-HCL pH 7.5, 0.5 Mammonium acetate, 10 mM EDTA, 0.1% SDS) at 37°C over-night or 65°C for 2 h. The RNAs were purified and concen-trated by an ammonium acetate precipitation andresuspended in 60 ml of DEPC-treated water. OD260 was usedto determine the concentration of RNAs.

Electrophoretic mobility shift assay (EMSA)

The purified in vitro synthesized DsrABb and rpoS RNAs wereincubated with increasing amounts of purified recombinantHfqBb in binding buffer (10 mM Tris-HCl, pH 7.5, 10 mMMgCl2, 50 mM NaCl and 50 mM KCl). Approximately 0.17pmoles of DsrABb or rpoS RNA was mixed with 7 ml of thebinding buffer, heated to 65°C for 10 min and slowly cooled toroom temperature. Two microlitres of serially diluted HfqBb

was added to the RNA and incubated for 5 min at roomtemperature. The samples were mixed with 10 ml of gelloading buffer II (Ambion), fractionated on a Novex® Pre-Cast



6% DNA retardation gel (Invitrogen) in 1¥ TBE at 100 V for2 h. The gel was dried, exposed to a phosphorimager screenand visualized using a Fujifilm FLA-3000G PhosphorImager.

Gene disruption and complementation of hfqBb

The hfqBb (bb0268) gene was deleted and replaced with akanamycin resistance cassette (Bono et al., 2000). Regionsflanking the hfqBb ORF were amplified by PCR (Table 2). The3′ end of the upstream flanking sequence and the 5′ end ofthe downstream flanking sequence were engineered withAatII sites for insertion of the kanamycin resistance cassette.The two flanking regions were cloned into pCR®2.1-TOPOand ligated together to form a 2.1 kb target DNA fragment.The antibiotic resistance cassette was cloned into the syn-thetic AatII site. The plasmid was linearized with AhdI andelectroporated into competent B. burgdorferi as previouslydescribed (Samuels, 1995). B. burgdorferi transformantswere cloned in liquid BSK-II medium (Yang et al., 2004) con-taining kanamycin (200 mg ml-1) at 34°C and a 1.5% CO2

atmosphere. Transformants were screened by PCR withprimers that flank the insertion site.

Two different plasmids were constructed to complement thehfqBb mutant strain. The E. coli or B. burgdorferi hfq genes werefused to the inducible flac promoter (Gilbert et al., 2007) andinserted into a modified pKFSS1 vector (Frank et al., 2003)carrying the lacI repressor gene fused to the constitutive flgBpromoter in the multiple cloning site. The recombinant plas-mids were electroporated into the kanamycin-resistant hfqBb

mutant and selected in both streptomycin and kanamycin. Theresulting transformants were assayed for the hfqBb expressionplasmid by both PCR and xenodiagnosis (transformation ofplasmid DNA from B. burgdorferi into E. coli ).

RT-PCR

Total RNA was isolated from 100 ml of cultures using TRIzol™(Gibco BRL) as previously described (Lybecker and Samuels,2007). RNA samples were treated with TurboDNase (Ambion)and RT-PCR was performed using the RETROscript™ kit(Ambion) according to manufacturer’s instructions, except agene-specific primer was used in the reverse transcriptionreaction instead of random decamers (Table 2).

Microscopic analysis of B. burgdorferi

Cell cultures were grown at 37°C to a low cell density(1–3 ¥ 107 cells ml-1) and centrifuged at 5800 g. Cell pelletswere resuspended in 100 ml of water. Samples were visual-ized by differential interference contrast (DIC) microscopyusing a Nikon E800 microscope at 100¥. B. burgdorferi lengthwas measured using ImageJ software. The length of hfqBb

mutant cells do not display a normal distribution, so signifi-cance was evaluated using both parametric (one-way ANOVA

with a Tukey’s post hoc test and a two-tailed unpaired t-test)and non-parametric (Kruskal–Wallis one-way ANOVA and aMann–Whitney U-test) methods.

SDS-PAGE and immunoblotting

Borrelia burgdorferi protein extracts were prepared as previ-ously described (Lybecker and Samuels, 2007). Protein

extracts were fractionated in Novex® Pre-Cast 4–20% Tris-Glycine gels (Invitrogen) and transferred to Immobilon-PPVDF membranes (Millipore) by electroblotting. Proteinswere detected with either an anti-RpoS antiserum (Yanget al., 2000) or anti-OspC antiserum (Yang et al., 2005). Themembranes were developed by chemiluminescence usingthe ECL Plus Western Blotting detection system (AmershamBiosciences) on a Fujifilm LAS-3000 (Yang et al., 2005;Gilbert et al., 2007).

Northern blot analysis

Total RNA was isolated from 100 ml of cultures usingTRIzol™ (Gibco BRL) as previously described (Lybecker andSamuels, 2007). RNA samples were treated with TurboD-Nase (Ambion) and fractionated in a Novex® Pre-Cast 6%polyacrylamide 7 M urea gel (Invitrogen) according to themanufacturers’ instructions. The RNA was transferred to aBrightStar-Plus™ membrane by electroblotting using theXcell SureLock™ Mini-Cell (Invitrogen) following the manu-facturer’s specifications. Northern blot hybridizations wereperformed using the NorthernMax™ kit (Ambion) accordingto the manufacturer’s instructions. Non-isotopic RNA probeswere generated from in vitro transcription of PCR-generatedDNA templates using the MAXIscript® in vitro transcription kit(Ambion). Biotin-16-UTP (Roche) was used in the in vitrotranscription reaction. Northern blot membranes were devel-oped by chemiluminescence using the BrightStar® Biode-tect™ non-isotopic detection kit on a Fujifilm LAS-3000.

Mouse infectivity studies

C3H-HeJ female mice were needle inoculated intraperito-neally with 5 ¥ 103 cells of wild-type 297 (BbAh130), hfqBb

mutant or complemented strains. All strains used in mouseinfectivity studies were screened for the presence of plasmidsessential for infectivity (lp28-1, lp25 and lp54) (Purser andNorris, 2000; Labandeira-Rey and Skare, 2001). Threeweeks post injection, ear punches were taken and cultured inBSK-II. Five weeks post injection, mice were sacrificed andear punches, tibiotarsal joints and bladders were collectedand cultivated in BSK-II. Cultures were screened for thepresence and growth of B. burgdorferi by dark-fieldmicroscopy. In addition, PCR was used to screen the culturesthat appeared negative by dark-field microscopy. The Univer-sity of Montana Institutional Animal Care and Use Committeeapproved all experimental procedures involving mice (proto-col number 038-08SSDBS-081208).

Acknowledgements

We thank Dan Drecktrah, Steve Lodmell and Jörg Vogel forthoughtful and critical reading of the manuscript; Dan Dreck-trah for help with image analysis; Jesse Hay for the use of hismicroscope; James Berger, Kevin Corbett, Mike Norgard,Craig Sampson (and the CDC Bacterial Zoonoses Branch),Darren Sledjeski, Philip Stewart and Frank Yang for strains,plasmids and/or antibodies; Dan Drecktrah, Jon Graham,Jean-Marc Lanchy, Steve Lodmell, Michele McGuirl and JörgVogel for useful discussions; the LAR staff for assistance with



animal experiments; Nilshad Salim and Tessa Samuels forlogistics; Patty McIntire (Murdock DNA Sequencing Facility)for DNA sequencing; and Laura Hall and Kelsey Lau forexcellent technical assistance. C.A.A. was supported by aWatkins Scholarship from The University of Montana, anUndergraduate Research Award from the Davidson HonorsCollege, a MILES Honors Fellowship through a grant from theHoward Hughes Medical Institute, and funding from the NSFEPSCoR Undergraduate Research Program. This work wassupported by grants from the National Institutes of Health(AI051486 to D.S.S. and GM075068 to A.L.F.).

References

Achsel, T., Brahms, H., Kastner, B., Bachi, A., Wilm, M., andLührmann, R. (1999) A doughnut-shaped heteromer ofhuman Sm-like proteins binds to the 3′-end of U6 snRNA,thereby facilitating U4/U6 duplex formation in vitro. EMBOJ 18: 5789–5802.

Achsel, T., Stark, H., and Lührmann, R. (2001) The Smdomain is an ancient RNA-binding motif with oligo(U)specificity. Proc Natl Acad Sci USA 98: 3685–3689.

Alverson, J., Bundle, S.F., Sohaskey, C.D., Lybecker, M.C.,and Samuels, D.S. (2003) Transcriptional regulation of theospAB and ospC promoters from Borrelia burgdorferi. MolMicrobiol 48: 1665–1677.

Antal, M., Bordeau, V., Douchin, V., and Felden, B. (2005) Asmall bacterial RNA regulates a putative ABC transporter.J Biol Chem 280: 7901–7908.

Arluison, V., Derreumaux, P., Allemand, F., Folichon, M.,Hajnsdorf, E., and Régnier, P. (2002) Structural modellingof the Sm-like protein Hfq from Escherichia coli. J Mol Biol320: 705–712.

Barbour, A.G. (1984) Isolation and cultivation of Lymedisease spirochetes. Yale J Biol Med 57: 521–525.

Barthold, S.W., Cadavid, D., and Philipp, M.T. (2010) Animalmodels of borreliosis. In Borrelia: Molecular Biology, HostInteraction and Pathogenesis. Samuels, D.S., and Radolf,J.D. (eds). Norfolk, UK: Caister Academic Press, pp. 359–411.

Benach, J.L., Bosler, E.M., Hanrahan, J.P., Coleman, J.L.,Bast, T.F., Habicht, G.S., et al. (1983) Spirochetes isolatedfrom the blood of two patients with Lyme disease. N Engl JMed 308: 740–742.

Blevins, J.S., Xu, H., He, M., Norgard, M.V., Reitzer, L., andYang, X.F. (2009) Rrp2, a s54-dependent transcriptionalactivator of Borrelia burgdorferi, activates rpoS in anenhancer-independent manner. J Bacteriol 191: 2902–2905.

Bono, J.L., Elias, A.F., Kupko, J.J., III, Stevenson, B., Tilly, K.,and Rosa, P. (2000) Efficient targeted mutagenesis in Bor-relia burgdorferi. J Bacteriol 182: 2445–2452.

Brennan, R.G., and Link, T.M. (2007) Hfq structure,function and ligand binding. Curr Opin Microbiol 10: 125–133.

Brescia, C.C., Mikulecky, P.J., Feig, A.L., and Sledjeski, D.D.(2003) Identification of the Hfq-binding site on DsrA RNA:Hfq binds without altering DsrA secondary structure. RNA9: 33–43.

Brown, L., and Elliott, T. (1997) Mutations that increaseexpression of the rpoS gene and decrease its dependence

on hfq function in Salmonella typhimurium. J Bacteriol 179:656–662.

Burgdorfer, W., Barbour, A.G., Hayes, S.F., Benach, J.L.,Grunwaldt, E., and Davis, J.P. (1982) Lyme disease – atick-borne spirochetosis? Science 216: 1317–1319.

Burtnick, M.N., Downey, J.S., Brett, P.J., Boylan, J.A., Frye,J.G., Hoover, T.R., and Gherardini, F.C. (2007) Insights intothe complex regulation of rpoS in Borrelia burgdorferi. MolMicrobiol 65: 277–293.

Caimano, M.J., Eggers, C.H., Hazlett, K.R., and Radolf, J.D.(2004) RpoS is not central to the general stress responsein Borrelia burgdorferi but does control expression of oneor more essential virulence determinants. Infect Immun 72:6433–6445.

Caimano, M.J., Eggers, C.H., Morton, E.A., Gilbert, M.A.,Schwartz, I., and Radolf, J.D. (2007) Analysis of the RpoSregulon in Borrelia burgdorferi in response to mammalianhost signals provides insight into RpoS function during theenzootic cycle. Mol Microbiol 65: 1193–1217.

Chao, Y., and Vogel, J. (2010) The role of Hfq in bacterialpathogens. Curr Opin Microbiol 13: 24–33.

Combet, C., Blanchet, C., Geourjon, C., and Deléage, G.(2000) NPS@: network protein sequence analysis. TrendsBiochem Sci 25: 147–150.

Corbett, K.D., Shultzaberger, R.K., and Berger, J.M. (2004)The C-terminal domain of DNA gyrase A adopts a DNA-bending b-pinwheel fold. Proc Natl Acad Sci USA 101:7293–7298.

Ding, Y., Davis, B.M., and Waldor, M.K. (2004) Hfq is essen-tial for Vibrio cholerae virulence and downregulates sE

expression. Mol Microbiol 53: 345–354.Fingerle, V., Laux, H., Munderloh, U.G., Schulte-Spechtel, U.,

and Wilske, B. (2000) Differential expression of outersurface proteins A and C by individual Borrelia burgdorferiin different genospecies. Med Microbiol Immunol 189:59–66.

Folichon, M., Arluison, V., Pellegrini, O., Huntzinger, E.,Régnier, P., and Hajnsdorf, E. (2003) The poly(A) bindingprotein Hfq protects RNA from RNase E and exoribonucle-olytic degradation. Nucleic Acids Res 31: 7302–7310.

Frank, K.L., Bundle, S.F., Kresge, M.E., Eggers, C.H., andSamuels, D.S. (2003) aadA confers streptomycin resis-tance in Borrelia burgdorferi. J Bacteriol 185: 6723–6727.

Franze de Fernandez, M.T., Eoyang, L., and August, J.T.(1968) Factor fraction required for the synthesis of bacte-riophage Qb-RNA. Nature 219: 588–590.

Fraser, C.M., Casjens, S., Huang, W.M., Sutton, G.G.,Clayton, R., Lathigra, R., et al. (1997) Genomic sequenceof a Lyme disease spirochete, Borrelia burgdorferi. Nature390: 580–586.

Fröhlich, K.S., and Vogel, J. (2009) Activation of gene expres-sion by small RNA. Curr Opin Microbiol 12: 674–682.

Geissmann, T.A., and Touati, D. (2004) Hfq, a new chaper-oning role: binding to messenger RNA determines accessfor small RNA regulator. EMBO J 23: 396–405.

Gilbert, M.A., Morton, E.A., Bundle, S.F., and Samuels, D.S.(2007) Artificial regulation of ospC expression in Borreliaburgdorferi. Mol Microbiol 63: 1259–1273.

Gottesman, S. (2004) The small RNA regulators of Escheri-chia coli: roles and mechanisms. Annu Rev Microbiol 58:303–328.



Hengge-Aronis, R. (2002) Signal transduction and regulatorymechanisms involved in control of the sS (RpoS) subunit ofRNA polymerase. Microbiol Mol Biol Rev 66: 373–395.

Hübner, A., Yang, X., Nolen, D.M., Popova, T.G., Cabello,F.C., and Norgard, M.V. (2001) Expression of Borrelia burg-dorferi OspC and DbpA is controlled by a RpoN–RpoSregulatory pathway. Proc Natl Acad Sci USA 98: 12724–12729.

Kambach, C., Walke, S., Young, R., Avis, J.M., Fortelle, E.,Raker, V.A., et al. (1999) Crystal structures of two Smprotein complexes and their implications for the assemblyof the spliceosomal snRNPs. Cell 96: 375–387.

Kawamoto, H., Koide, Y., Morita, T., and Aiba, H. (2006)Base-pairing requirement for RNA silencing by a bacterialsmall RNA and acceleration of duplex formation by Hfq.Mol Microbiol 61: 1013–1022.

Kelley, L.A., and Sternberg, M.J. (2009) Protein structureprediction on the Web: a case study using the Phyreserver. Nat Protoc 4: 363–371.

Klinkert, B., and Narberhaus, F. (2009) Microbialthermosensors. Cell Mol Life Sci 66: 2661–2676.

Labandeira-Rey, M., and Skare, J.T. (2001) Decreased infec-tivity in Borrelia burgdorferi strain B31 is associated withloss of linear plasmid 25 or 28-1. Infect Immun 69: 446–455.

Lane, R.S., Piesman, J., and Burgdorfer, W. (1991) Lymeborreliosis: relation of its causative agent to its vectors andhosts in North America and Europe. Annu Rev Entomol 36:587–609.

Lease, R.A., and Belfort, M. (2000) Riboregulation by DsrARNA: trans-actions for global economy. Mol Microbiol 38:667–672.

Lease, R.A., and Woodson, S.A. (2004) Cycling of theSm-like protein Hfq on the DsrA small regulatory RNA.J Mol Biol 344: 1211–1223.

Lee, T., and Feig, A.L. (2008) The RNA binding protein Hfqinteracts specifically with tRNAs. RNA 14: 514–523.

Link, T.M., Valentin-Hansen, P., and Brennan, R.G. (2009)Structure of Escherichia coli Hfq bound to polyriboadeny-late RNA. Proc Natl Acad Sci USA 106: 19292–19297.

Lybecker, M.C., and Samuels, D.S. (2007) Temperature-induced regulation of RpoS by a small RNA in Borreliaburgdorferi. Mol Microbiol 64: 1075–1089.

Majdalani, N., Vanderpool, C.K., and Gottesman, S. (2005)Bacterial small RNA regulators. Crit Rev Biochem Mol Biol40: 93–113.

Massé, E., and Gottesman, S. (2002) A small RNA regulatesthe expression of genes involved in iron metabolism inEscherichia coli. Proc Natl Acad Sci USA 99: 4620–4625.

Massé, E., Majdalani, N., and Gottesman, S. (2003a) Regu-latory roles for small RNAs in bacteria. Curr Opin Microbiol6: 120–124.

Massé, E., Escorcia, F.E., and Gottesman, S. (2003b)Coupled degradation of a small regulatory RNA and itsmRNA targets in Escherichia coli. Genes Dev 17: 2374–2383.

Mikulecky, P.J., Kaw, M.K., Brescia, C.C., Takach, J.C.,Sledjeski, D.D., and Feig, A.L. (2004) Escherichia coli Hfqhas distinct interaction surfaces for DsrA, rpoS and poly(A)RNAs. Nat Struct Mol Biol 11: 1206–1214.

Miller, J.H. (1972) Experiments in Molecular Genetics. ColdSpring Harbor, NY: Cold Spring Harbor Laboratory.

Moll, I., Leitsch, D., Steinhauser, T., and Bläsi, U. (2003a)RNA chaperone activity of the Sm-like Hfq protein. EMBORep 4: 284–289.

Moll, I., Afonyushkin, T., Vytvytska, O., Kaberdin, V.R., andBläsi, U. (2003b) Coincident Hfq binding and RNase Ecleavage sites on mRNA and small regulatory RNAs. RNA9: 1308–1314.

Møller, T., Franch, T., Højrup, P., Keene, D.R., Bächinger,H.P., Brennan, R.G., and Valentin-Hansen, P. (2002) Hfq: abacterial Sm-like protein that mediates RNA–RNAinteraction. Mol Cell 9: 23–30.

Moskaleva, O., Melnik, B., Gabdulkhakov, A., Garber, M.,Nikonov, S., Stolboushkina, E., and Nikulin, A. (2010) Thestructures of mutant forms of Hfq from Pseudomonasaeruginosa reveal the importance of the conserved His57for the protein hexamer organization. Acta CrystallographSect F Struct Biol Cryst Commun 66: 760–764.

Muffler, A., Traulsen, D.D., Lange, R., and Hengge-Aronis, R.(1996) Posttranscriptional osmotic regulation of the sS

subunit of RNA polymerase in Escherichia coli. J Bacteriol178: 1607–1613.

Muffler, A., Traulsen, D.D., Fischer, D., Lange, R., andHengge-Aronis, R. (1997) The RNA-binding protein HF-Iplays a global regulatory role which is largely, but notexclusively, due to its role in expression of the sS subunit ofRNA polymerase in Escherichia coli. J Bacteriol 179: 297–300.

Narberhaus, F., Waldminghaus, T., and Chowdhury, S.(2006) RNA thermometers. FEMS Microbiol Rev 30: 3–16.

Nielsen, J.S., Bøggild, A., Andersen, C.B.F., Nielsen, G.,Boysen, A., Brodersen, D.E., and Valentin-Hansen, P.(2007) An Hfq-like protein in archaea: crystal structure andfunctional characterization of the Sm protein from Metha-nococcus jannaschii. RNA 13: 2213–2223.

Ojaimi, C., Brooks, C., Casjens, S., Rosa, P., Elias, A.,Barbour, A., et al. (2003) Profiling of temperature-inducedchanges in Borrelia burgdorferi gene expression by usingwhole genome arrays. Infect Immun 71: 1689–1705.

Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S.,Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004)UCSF Chimera – a visualization system for exploratoryresearch and analysis. J Comput Chem 25: 1605–1612.

Piesman, J., and Schwan, T.G. (2010) Ecology of borreliaeand their arthropod vectors. In Borrelia: Molecular Biology,Host Interaction and Pathogenesis. Samuels, D.S., andRadolf, J.D. (eds). Norfolk, UK: Caister Academic Press,pp. 251–278.

Purser, J.E., and Norris, S.J. (2000) Correlation betweenplasmid content and infectivity in Borrelia burgdorferi. ProcNatl Acad Sci USA 97: 13865–13870.

Radolf, J.D., Salazar, J.C., and Dattwyler, R.J. (2010) Lymedisease in humans. In Borrelia: Molecular Biology, HostInteraction and Pathogenesis. Samuels, D.S., and Radolf,J.D. (eds). Norfolk, UK: Caister Academic Press, pp. 487–533.

Repoila, F., Majdalani, N., and Gottesman, S. (2003) Smallnon-coding RNAs, co-ordinators of adaptation processesin Escherichia coli: the RpoS paradigm. Mol Microbiol 48:855–861.

Revel, A.T., Talaat, A.M., and Norgard, M.V. (2002) DNAmicroarray analysis of differential gene expression in Bor-



relia burgdorferi, the Lyme disease spirochete. Proc NatlAcad Sci USA 99: 1562–1567.

Robertson, G.T., and Roop, R.M., Jr (1999) The Brucellaabortus host factor I (HF-I) protein contributes to stressresistance during stationary phase and is a major determi-nant of virulence in mice. Mol Microbiol 34: 690–700.

Romby, P., Vandenesch, F., and Wagner, E.G. (2006) Therole of RNAs in the regulation of virulence-geneexpression. Curr Opin Microbiol 9: 229–236.

Samuels, D.S. (1995) Electrotransformation of the spirocheteBorrelia burgdorferi. In Electroporation Protocols forMicroorganisms. Nickoloff, J.A. (ed.). Totowa, NJ: HumanaPress, pp. 253–259.

Samuels, D.S., and Radolf, J.D. (2009) Who is the BosRaround here anyway? Mol Microbiol 74: 1295–1299.

Sauter, C., Basquin, J., and Suck, D. (2003) Sm-like proteinsin Eubacteria: the crystal structure of the Hfq protein fromEscherichia coli. Nucleic Acids Res 31: 4091–4098.

Schumacher, M.A., Pearson, R.F., Møller, T., Valentin-Hansen, P., and Brennan, R.G. (2002) Structures of thepleiotropic translational regulator Hfq and an Hfq–RNAcomplex: a bacterial Sm-like protein. EMBO J 21: 3546–3556.

Schwan, T.G., Piesman, J., Golde, W.T., Dolan, M.C., andRosa, P.A. (1995) Induction of an outer surface protein onBorrelia burgdorferi during tick feeding. Proc Natl Acad SciUSA 92: 2909–2913.

Serganov, A., and Patel, D.J. (2007) Ribozymes,riboswitches and beyond: regulation of gene expressionwithout proteins. Nat Rev Genet 8: 776–790.

Singh, S.K., and Girschick, H.J. (2004) Molecular survivalstrategies of the Lyme disease spirochete Borreliaburgdorferi. Lancet Infect Dis 4: 575–583.

Sittka, A., Pfeiffer, V., Tedin, K., and Vogel, J. (2007) TheRNA chaperone Hfq is essential for the virulence of Sal-monella typhimurium. Mol Microbiol 63: 193–217.

Skare, J.T., Carroll, J.A., Yang, X.F., Samuels, D.S., andAkins, D.R. (2010) Gene regulation, transcriptomics andproteomics. In Borrelia: Molecular Biology, Host Interactionand Pathogenesis. Samuels, D.S., and Radolf, J.D. (eds).Norfolk, UK: Caister Academic Press, pp. 67–101.

Sledjeski, D.D., Whitman, C., and Zhang, A. (2001) Hfq isnecessary for regulation by the untranslated RNA DsrA.J Bacteriol 183: 1997–2005.

Spielman, A. (1994) The emergence of Lyme disease andhuman babesiosis in a changing environment. Ann N YAcad Sci 740: 146–156.

Steere, A.C., Grodzicki, R.L., Kornblatt, A.N., Craft, J.E.,Barbour, A.G., Burgdorfer, W., et al. (1983) The spirochetaletiology of Lyme disease. N Engl J Med 308: 733–740.

Stevenson, B., Schwan, T.G., and Rosa, P.A. (1995)Temperature-related differential expression of antigens inthe Lyme disease spirochete, Borrelia burgdorferi. InfectImmun 63: 4535–4539.

Storz, G., Opdyke, J.A., and Zhang, A. (2004) ControllingmRNA stability and translation with small, noncodingRNAs. Curr Opin Microbiol 7: 140–144.

Sun, X., Zhulin, I., and Wartell, R.M. (2002) Predicted struc-ture and phyletic distribution of the RNA-binding proteinHfq. Nucleic Acids Res 30: 3662–3671.

Törö, I., Thore, S., Mayer, C., Basquin, J., Séraphin, B., andSuck, D. (2001) RNA binding in an Sm core domain: X-raystructure and functional analysis of an archaeal Sm proteincomplex. EMBO J 20: 2293–2303.

Törö, I., Basquin, J., Teo-Dreher, H., and Suck, D. (2002)Archaeal Sm proteins form heptameric and hexamericcomplexes: crystal structures of the Sm1 and Sm2 proteinsfrom the hyperthermophile Archaeoglobus fulgidus. J MolBiol 320: 129–142.

Tsui, H.-C.T., Leung, H.C., and Winkler, M.E. (1994) Charac-terization of broadly pleiotropic phenotypes caused by anhfq insertion mutation in Escherichia coli K-12. Mol Micro-biol 13: 35–49.

Tsui, H.-C.T., Feng, G., and Winkler, M.E. (1997) Negativeregulation of mutS and mutH repair gene expression by theHfq and RpoS global regulators of Escherichia coli K-12.J Bacteriol 179: 7476–7487.

Valentin-Hansen, P., Eriksen, M., and Udesen, C. (2004) Thebacterial Sm-like protein Hfq: a key player in RNAtransactions. Mol Microbiol 51: 1525–1533.

Vecerek, B., Moll, I., and Bläsi, U. (2005) Translational auto-control of the Escherichia coli hfq RNA chaperone gene.RNA 11: 976–984.

Vecerek, B., Moll, I., and Bläsi, U. (2007) Control of Fursynthesis by the non-coding RNA RyhB and iron-responsive decoding. EMBO J 26: 965–975.

Vytvytska, O., Moll, I., Kaberdin, V.R., von Gabain, A., andBläsi, U. (2000) Hfq (HF1) stimulates ompA mRNA decayby interfering with ribosome binding. Genes Dev 14: 1109–1118.

Wang, G., Aguero-Rosenfeld, M.E., Wormser, G.P., andSchwartz, I. (2010) Detection of Borrelia burgdorferi. InBorrelia: Molecular Biology, Host Interaction andPathogenesis. Samuels, D.S., and Radolf, J.D. (eds).Norfolk, UK: Caister Academic Press, pp. 443–466.

Waters, L.S., and Storz, G. (2009) Regulatory RNAs inbacteria. Cell 136: 615–628.

Yang, X., Goldberg, M.S., Popova, T.G., Schoeler, G.B.,Wikel, S.K., Hagman, K.E., and Norgard, M.V. (2000) Inter-dependence of environmental factors influencing recipro-cal patterns of gene expression in virulent Borreliaburgdorferi. Mol Microbiol 37: 1470–1479.

Yang, X.F., Alani, S.M., and Norgard, M.V. (2003) Theresponse regulator Rrp2 is essential for the expression ofmajor membrane lipoproteins in Borrelia burgdorferi. ProcNatl Acad Sci USA 100: 11001–11006.

Yang, X.F., Pal, U., Alani, S.M., Fikrig, E., and Norgard, M.V.(2004) Essential role for OspA/B in the life cycle of theLyme disease spirochete. J Exp Med 199: 641–648.

Yang, X.F., Lybecker, M.C., Pal, U., Alani, S.M., Blevins, J.,Revel, A.T., et al. (2005) Analysis of the ospC regulatoryelement controlled by the RpoN–RpoS regulatory pathwayin Borrelia burgdorferi. J Bacteriol 187: 4822–4829.

Zhang, A., Wassarman, K.M., Ortega, J., Steven, A.C., andStorz, G. (2002) The Sm-like Hfq protein increases OxySRNA interaction with target mRNAs. Mol Cell 9: 11–22.

Zhang, X., and Bremer, H. (1995) Control of the Escherichiacoli rrnB P1 promoter strength by ppGpp. J Biol Chem 270:11181–11189.

Zuker, M. (2003) Mfold web server for nucleic acid foldingand hybridization prediction. Nucleic Acids Res 31: 1–10.



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