Hfq Mutant Research Articles

Shewanella oneidensisHfq promotes exponential phase growth, stationary phase culture density, and cell survival

BackgroundHfq is an RNA chaperone protein that has been broadly implicated in sRNA function in bacteria. Here we describe the construction and characterization of a null allele of the gene that encodes the RNA chaperone Hfq in Shewanella oneidensis strain MR-1, a dissimilatory metal reducing bacterium.ResultsLoss of hfq in S. oneidensis results in a variety of mutant phenotypes, all of which are fully complemented by addition of a plasmid-borne copy of the wild type hfq gene. Aerobic cultures of the hfq∆ mutant grow more slowly through exponential phase than wild type cultures, and hfq∆ cultures reach a terminal cell density in stationary phase that is ~2/3 of that observed in wild type cultures. We have observed a similar growth phenotype when the hfq∆ mutant is cultured under anaerobic conditions with fumarate as the terminal electron acceptor, and we have found that the hfq∆ mutant is defective in Cr(VI) reduction. Finally, the hfq∆ mutant exhibits a striking loss of colony forming units in extended stationary phase and is highly sensitive to oxidative stress induced by H2O2 or methyl viologen (paraquat).ConclusionsThe hfq mutant in S. oneidensis exhibits pleiotropic phenotypes, including a defect in metal reduction. Our results also suggest that hfq mutant phenotypes in S. oneidensis may be at least partially due to increased sensitivity to oxidative stress.

Mutations in Interaction Surfaces Differentially Impact E. coli Hfq Association with Small RNAs and Their mRNA Targets

The RNA chaperone protein Hfq is required for the function of all small RNAs (sRNAs) that regulate mRNA stability or translation by limited base pairing in Escherichia coli. While there have been numerous in vitro studies to characterize Hfq activity and the importance of specific residues, there has been only limited characterization of Hfq mutants in vivo. Here, we use a set of reporters as well as co-immunoprecipitation to examine 14 Hfq mutants expressed from the E. coli chromosome. The majority of the proximal face residues, as expected, were important for the function of sRNAs. The failure of sRNAs to regulate target mRNAs in these mutants can be explained by reduced sRNA accumulation. Two of the proximal mutants, D9A and F39A, acted differently from the others in that they had mixed effects on different sRNA/mRNA pairs and, in the case of F39A, showed differential sRNA accumulation. Mutations of charged residues at the rim of Hfq interfered with positive regulation and gave mixed effects for negative regulation. Some, but not all, sRNAs accumulated to lower levels in rim mutants, suggesting qualitative differences in how individual sRNAs are affected by Hfq. The distal face mutants were expected to disrupt binding of ARN motifs found in mRNAs. They were more defective for positive regulation than negative regulation at low mRNA expression, but the defects could be suppressed by higher levels of mRNA expression. We discuss the implications of these observations for Hfq binding to RNA and mechanisms of action.

Quantitative Proteomic Analysis of the Hfq-Regulon in Sinorhizobium meliloti 2011

Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with 15N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti.

Identification of two small regulatory RNAs linked to virulence in Brucella abortus 2308

Hfq is an RNA-binding protein that functions in post-transcriptional gene regulation by mediating interactions between mRNAs and small regulatory RNAs (sRNAs). Two proteins encoded by BAB1_1794 and BAB2_0612 are highly over-produced in a Brucella abortus hfq mutant compared with the parental strain, and recently, expression of orthologues of these proteins in Agrobacterium tumefaciens was shown to be regulated by two sRNAs, called AbcR1 and AbcR2. Orthologous sRNAs (likewise designated AbcR1 and AbcR2) have been identified in B. abortus 2308. In Brucella, abcR1 and abcR2 single mutants are not defective in their ability to survive in cultured murine macrophages, but an abcR1 abcR2 double mutant exhibits significant attenuation in macrophages. Additionally, the abcR1 abcR2 double mutant displays significant attenuation in a mouse model of chronic Brucella infection. Quantitative proteomics and microarray analyses revealed that the AbcR sRNAs predominantly regulate genes predicted to be involved in amino acid and polyamine transport and metabolism, and Northern blot analyses indicate that the AbcR sRNAs accelerate the degradation of the target mRNAs. In an Escherichia coli two-plasmid reporter system, overexpression of either AbcR1 or AbcR2 was sufficient for regulation of target mRNAs, indicating that the AbcR sRNAs from B. abortus 2308 perform redundant regulatory functions.

Construction and characterization of a null allele of the hfq gene of the dissimilatory metal reducing bacterium Shewanella oneidensis

Shewanella oneidensis is a dissimilatory metal reducing bacterium that can utilize a range of terminal electron acceptors when grown under anaerobic conditions. Our primary goal is to identify and characterize genes that encode small regulatory non‐coding RNAs (sRNAs) in S. oneidensis. sRNA genes are regulated by changes in environmental conditions and can themselves regulate other genes by inexact base pairing to their mRNA targets. We are particularly interested in sRNAs that may regulate the process of metal reduction.The highly conserved RNA chaperone protein Hfq has been widely implicated in bacterial sRNA function. We have constructed a null allele of the S. oneidensis hfq gene. The S. oneidensis hfq mutant has a modest delayed growth phenotype. In addition, our mutant displays reduced survival in stationary phase. Our hfq mutant also exhibits a decreased rate of Cr(VI) reduction when grown under anaerobic conditions compared to the rate of Cr(VI) reduction by wild type S. oneidensis. We are currently evaluating the growth and survival of the Shewanella hfq null mutant. We are also investigating the mutant's sensitivity to a variety of physical stresses, evaluating the capacity of the hfq mutant to reduce metals other than Cr(VI), and determining the role of Shewanella hfq in sRNA function.This research is funded by a grant from the RI‐INBRE program, award # P20RR016457‐10.

Hfq and Hfq-dependent small RNAs are major contributors to multicellular development in Salmonella enterica serovar Typhimurium

The RNA chaperone Hfq and its associated small RNAs (sRNAs) regulate a variety of phenotypes in bacteria. In this work, we show that Hfq is a master regulator of biofilm formation in Salmonella enterica serovar Typhimurium. Hfq and two Hfq-dependent sRNAs (ArcZ and SdsR) are required for rdar morphotype expression in S. typhimurium. Hfq controls rdar biofilm formation through the major biofilm regulator CsgD. While csgD mRNA steady-state levels are altered in a sdsR mutant, ArcZ seems to work mainly at the post-transcriptional level. Overexpression of ArcZ complemented rdar morphotype formation of an hfq mutant under plate-grown conditions. Although ArcZ activates rpoS expression, its effect on csgD expression is mainly independent of RpoS. ArcZ does not only regulate rdar morphotype expression, but also the transition between sessility and motility and the timing of type 1 fimbriae vs. curli fimbriae surface-attachment at ambient temperature. Consequently, ArcZ is a major regulator of rdar biofilm development.

Impact of Hfq on the Intrinsic Drug Resistance of Salmonella Enterica Serovar Typhimurium

Salmonella enterica is an important enteric pathogen, and its various serovars cause both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella, leading to increased morbidity and mortality, has further complicated its management. Hfq is an RNA chaperon that mediates the binding of small RNAs to mRNA and assists in post-transcriptional gene regulation in bacteria. Although Hfq is related to important phenotypes including virulence in Salmonella, its role in the drug resistance of this organism is unknown. The aim of this study was to investigate the role of Hfq in intrinsic drug resistance of S. enterica serovar Typhimurium. hfq Mutant was susceptible to acriflavine. Although there is a relationship between the production of the AcrB multidrug efflux pump and Hfq in Escherichia coli, the deletion of the drug efflux acrB did not impair the effect of hfq deletion on Salmonella susceptibility. In contrast, the deletion of another drug efflux gene, smvA, impaired the effect of hfq deletion on acriflavine susceptibility. These results indicate that Hfq regulates the intrinsic drug resistance, and it may influence drug susceptibility by regulating SmvA in Salmonella.

The RNA Chaperone Hfq Independently Coordinates Expression of the VirB Type IV Secretion System and the LuxR-Type Regulator BabR in Brucella abortus 2308

The type IV secretion system encoded by the virB operon is required for full virulence of Brucella sp., and the present study links the RNA chaperone Hfq to wild-type expression of virB in Brucella abortus 2308. Studies employing virB-lacZ fusions, quantitative reverse transcription-PCR, and immunoblot analysis showed that both transcription and translation of virB are decreased in an isogenic hfq mutant compared to those in the parental strain. These results led to the hypothesis that Hfq regulation of virB is mediated through an intermediate transcriptional regulator. Subsequent experiments determined that expression of the gene encoding the putative Brucella quorum-sensing regulator BabR (also known as BlxR), a known virB regulator, is also controlled by Hfq at the posttranscriptional level, and a cis-acting element in the 5' untranslated region of the babR transcript responsible for this regulation was identified. Consistent with its role as a virB regulator, recombinant Brucella BabR binds to the virB promoter region in electrophoretic mobility shift assays. However, experiments employing a babR mutant strain determined that BabR is a repressor, not an activator, of virB transcription. These findings suggest that Hfq regulates virB expression through both BabR-dependent and BabR-independent pathways.

Hfq Is Required for Optimal Nitrate Assimilation in the Cyanobacterium Anabaena sp. Strain PCC 7120

Hfq is an RNA binding protein involved in posttranscriptional regulation of gene expression in bacteria. It acts by binding to regulatory small RNAs (sRNAs), which confer specificity for the regulation. Recently, orthologues of the Hfq protein were annotated in cyanobacterial genomes, although its capacity to regulate gene expression by interacting with sRNAs has not been yet demonstrated. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that, in the absence of combined nitrogen, is able to fix atmospheric nitrogen by differentiating specialized cells called heterocysts. We have generated an hfq knockout mutant of Anabaena sp. PCC 7120. Deletion of this gene results in differentiation of heterocysts in the presence of nitrate, suggesting a defect in nitrate assimilation. We show that hfq mutant cells are affected in transport and use of nitrate and nitrite. An analysis of the expression of several genes in the nir operon, encoding different elements of the nitrate assimilation pathway, demonstrates a downregulation of their transcription in mutant cells. We also observed that genes ntcB and cnaT, involved in the regulation of the nir operon, show a lower expression in cells lacking Hfq. Finally, when hfq was reintroduced in the mutant, heterocyst differentiation was no longer observed in the presence of nitrate. Therefore, our results indicate that the RNA chaperone Hfq is involved in the regulation of the nir operon, although the mechanism for this regulation is still unknown.

The RNA chaperone Hfq is important for growth and stress tolerance in Francisella novicida.

The RNA-binding protein Hfq is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis. Hfq has been shown in several bacteria to interact with small regulatory RNAs and act as a post-transcriptional regulator of mRNA stability and translation. Here we examined the impact of Hfq on growth, stress tolerance, and gene expression in the intracellular pathogen Francisella novicida. We present evidence of Hfq involvement in the ability of F. novicida to tolerate several cellular stresses, including heat-shock and oxidative stresses, and alterations in hfq gene expression under these conditions. Furthermore, expression of numerous genes, including several associated with virulence, is altered in a hfq mutant strain suggesting they are regulated directly or indirectly by Hfq. Strikingly, we observed a delayed entry into stationary phase and increased biofilm formation in the hfq mutant. Together, these data demonstrate a critical role for Hfq in F. novicida growth and survival.

Despite Similar Binding to the Hfq Protein Regulatory RNAs Widely Differ in Their Competition Performance

The binding of nine noncoding regulatory RNAs (sRNAs) to the E. coli Hfq protein was compared using a high-throughput double filter retention assay. Despite the fact that these sRNAs have different lengths, sequences and secondary structures their Hfq binding affinities were surprisingly uniform. The analysis of sRNAs binding to Hfq mutants showed that the proximal face of Hfq, known as the binding site for DsrA RNA, is a universal sRNA binding site. Moreover, all sRNAs bound Hfq with similar association rates limited only by the rate of diffusion, while the rates of dissociation, measured in the dilution experiments, were uniformly slow. Despite that, the data showed that there was a hierarchy of sRNAs in regard to their performance in competition for access to Hfq and in their ability to facilitate the dissociation of other sRNAs from Hfq. The sRNAs also differed in their salt dependence of binding to this protein. Overall, the results suggest that despite the uniform binding of different sRNAs to the same site on Hfq their exchange on this protein is dependent on the identities of the competing sRNAs.

Role of polynucleotide phosphorylase in sRNA function in Escherichia coli

In Escherichia coli, many small noncoding regulatory RNAs (sRNAs) post-transcriptionally regulate gene expression by base-pairing to mRNAs in a process that is mediated by the RNA chaperone Hfq. Binding of the sRNA to the mRNA can lead to increased or decreased mRNA stability and/or translation. It is not known if proteins other than Hfq are necessary for this process. In order to identify additional genes required for the post-transcriptional regulation of gene expression by Hfq-dependent sRNAs, we developed a novel combined genetic selection and screen for mutants defective in sRNA regulation. In our combined genetic selection and screen, we isolated hfq mutants and mutants in pnp, encoding polynucleotide phosphorylase (PNPase). We show that loss-of-function mutations in pnp result in a decreased stability of several sRNAs including RyhB, SgrS, and CyaR and also decrease both the negative and positive regulation by sRNAs. The defect in stability of CyaR and in negative and positive regulation are suppressed by deletion mutations in RNase E. Altogether, our results suggest that the lack of sRNA-mediated regulation in the absence of an active form of PNPase is due to the rapid turnover of sRNA resulting from an increase in RNase E activity and/or an increase in access of other ribonucleases to sRNAs.

Roles of Hfq in the stress adaptation and virulence in fish pathogen Vibrio alginolyticus and its potential application as a target for live attenuated vaccine

Vibrio alginolyticus has brought about severe economic damage to the mariculture industry by causing vibriosis in various fish species in South China. The virulent determinants of this bacterium have not been well characterized except the exotoxin alkaline serine protease, Asp. In addition, the mechanism of virulence regulation in V. alginolyticus remains largely unknown apart from a Vibrio harveyi-like quorum sensing (QS) system which is established to manipulate the expression of various virulence-related genes. Hfq, an sRNA chaperone, is an important post-transcriptional regulator in a variety of bacteria. Here, the roles of Hfq were characterized in regulating the stress resistance and pathogenesis in V. alginolyticus. We demonstrated that the hfq deletion mutant became more sensitive to several environmental stresses, including osmotic stress, ethanol, temperature shift, and iron starvation. The deletion of hfq abrogated the motility and biofilm formation in this bacterium. Hfq negatively regulated the expression of main virulence factor, Asp, through QS system. The results also indicated that Hfq modulated the survival and multiplication of V. alginolyticus in fish. Hfq thus appears to be a new pleiotropic regulator of pathogenesis in V. alginolyticus. Moreover, high immunoprotective rate was achieved with a single dose of injection or immersion vaccination with live hfq mutant, suggesting the mutant's merits as a valuable vaccine candidate against V. alginolyticus.

A novel Hfq‐dependent sRNA that is under FNR control and is synthesized in oxygen limitation in Neisseria meningitidis

Small non-coding RNAs (sRNA) are emerging as key elements of post-transcriptional gene regulation in bacteria. The conserved Hfq protein is thought to function as an RNA chaperone and facilitate base-pairing between sRNAs and mRNA targets. In this study we identify a novel sRNA of Neisseria meningitidis through global gene expression studies of regulated transcripts in the Hfq mutant. The synthesis of this sRNA, named AniS, is anaerobically induced through activation of its promoter by the FNR global regulator. Whole-genome expression analyses led to the identification of putative mRNA targets, two of which are predicted to base pair with AniS. We show that Hfq binds the AniS transcript in vitro and is necessary for the downregulation of the identified target mRNAs in vivo. Contrary to many Hfq-dependent sRNA of the Enterobacteriaceae, Hfq promotes decay of AniS in N. meningitidis. Our analysis shows that the AniS regulator is part of the FNR regulon and may be responsible for the downregulation of FNR-repressed genes. Furthermore the presence of similar conserved regulatory sequences in all Neisseria spp. to date suggests that an analogous FNR-regulated sRNA, with a variable 5' sequence, may be ubiquitous to all commensals and pathogens of the Genus.

Mutation of GOGAT prevents pea bacteroid formation and N2 fixation by globally downregulating transport of organic nitrogen sources.

Mutation of gltB (encoding glutamate oxoglutarate amidotransferase or GOGAT) in RU2307 increased the intracellular Gln:Glu ratio and inhibited amino acid transport via Aap and Bra. The mechanism probably involves global post-translational inhibition independent of Ntr. Transport was separately restored by increased gene expression of Aap or heterologous transporters. Likewise, second site suppressor mutations in the RNA chaperone Hfq elevated transport by Aap and Bra by increasing mRNA levels. Microarrays showed Hfq regulates 34 ABC transporter genes, including aap, bra and opp. The genes coding for integral membrane proteins and ABC subunits aapQMP braDEFGC were more strongly elevated in the hfq mutants than solute-binding proteins (aapJ braC). aapQMP and braDEFG are immediately downstream of stem-loops, indicating Hfq attenuates downstream translation and stability of mRNA, explaining differential expression of ABC genes. RU2307 nodulated peas and bacteria grew down infection threads, but bacteroid development was arrested and N(2) was not fixed. This probably results from an inability to synthesize or transport amino acids. However, GOGAT and GOGAT/AldA double mutants carrying suppressor mutations that increased amino acid uptake fixed N(2) on pea plants. Thus de novo ammonium assimilation into amino acids is unnecessary in bacteroids demonstrating sufficient amino acids are supplied by plants.

A highly conserved protein of unknown function in Sinorhizobium meliloti affects sRNA regulation similar to Hfq

The SMc01113/YbeY protein, belonging to the UPF0054 family, is highly conserved in nearly every bacterium. However, the function of these proteins still remains elusive. Our results show that SMc01113/YbeY proteins share structural similarities with the MID domain of the Argonaute (AGO) proteins, and might similarly bind to a small-RNA (sRNA) seed, making a special interaction with the phosphate on the 5′-side of the seed, suggesting they may form a component of the bacterial sRNA pathway. Indeed, eliminating SMc01113/YbeY expression in Sinorhizobium meliloti produces symbiotic and physiological phenotypes strikingly similar to those of the hfq mutant. Hfq, an RNA chaperone, is central to bacterial sRNA-pathway. We evaluated the expression of 13 target genes in the smc01113 and hfq mutants. Further, we predicted the sRNAs that may potentially target these genes, and evaluated the accumulation of nine sRNAs in WT and smc01113 and hfq mutants. Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs. AGOs are central components of the eukaryotic sRNA machinery and conceptual parallels between the prokaryotic and eukaryotic sRNA pathways have long been drawn. Our study provides the first line of evidence for such conceptual parallels. Furthermore, our investigation gives insights into the sRNA-mediated regulation of stress adaptation in S. meliloti.

The Role of Sm-Like Protein Hfq in RNA Annealing

Small RNAs (sRNAs) regulate bacterial response and adaptation to environmental stress. This RNA regulation mechanism requires the Sm-like binding protein, Hfq, which promotes annealing of complimentary RNAs. The distal face of Hfq interacts with poly(A) sequences, while the proximal face interacts with poly(U) sequences. The sRNA DsrA binds to the rpoS mRNA, opening an inhibitory stem loop in the mRNA, and consequently initiating translation of the RpoS sigma stress response factor in Escherichia coli. Hfq binding to an (AAN)4 repeat element upstream of the inhibitor stem loop on rpoS mRNA has been shown to be required to promote DsrA annealing. To further understand the role of Hfq in RNA annealing, we studied the formation of the ternary complex between Hfq, sRNA DsrA, and mRNA rpoS mRNA. Two mutations were chosen to disrupt RNA binding to either the proximal or distal face of the Hfq hexamer. Fluorescence Anisotropy experiments were used to study the binding affinity of Hfq mutants to poly(U) oligomers. Y25D, on the distal face of Hfq, showed only a two fold decrease in binding affinity; whereas K56A, on the proximal face, showed a 5-fold decrease. Native gel mobility shift assays showed that only the K56A mutant formed a ternary complex with DsrA and rpoS RNAs. In contrast, the Y25D mutation inhibits rpoS mRNA binding and also fails to form a ternary complex. These results support previous results showing that Hfq binding to A-rich sequences in rpoS mRNA is critical for rpoS regulation.

Computational approach to ensure the stability of the favorable ATP binding site in E. coli Hfq

Bacterial Hfq is a highly conserved thermostable protein of about 10 kDa. The Hfq protein was discovered in 1968 as an E. coli host factor that was essential for replication of the bacteriophage Qβ. It is now clear that Hfq has many important physiological roles. In E. coli, Hfq mutants show a multiple stress response related phenotypes. Hfq is now known to regulate the translation of two major stress transcription factors RpoS and RpoE in Enterobacteria and mediates its plieotrophic effects through several mechanisms. It interacts with regulatory sRNA and facilitates their antisense interaction with their targets. It also acts independently to modulate mRNA decay and in addition acts as a repressor of mRNA translation. Recent paper from Arluison et al. [9] provided the first evidence indicating that Hfq is an ATP-binding protein. They determined a plausible ATP-binding site in Hfq and tested Hfq's ATP-binding affinity and stoichiometry. Experimental data suggest that the ATP-binding by the Hfq–RNA complex results in its significant destabilization of the protein and the result also proves important role of Tyr25 that flanks the cleft and stabilizes the adenine portion of ATP, possibly via aromatic stacking. In our study, the ATP molecule was docked into the predicted binding cleft using GOLD docking software. The binding nature of ATP and its effect on Hfq–RNA complex was studied using molecular dynamics simulations. Importance of Tyr25 residue was monitored and revealed using mutational study on the modeled systems. Our data and the corresponding results point to one of Hfq functional structural consequences due to ATP binding and Tyr25Ala mutation.

Hfq Is a Global Regulator That Controls the Pathogenicity of Staphylococcus aureus

The Hfq protein is reported to be an RNA chaperone, which is involved in the stress response and the virulence of several pathogens. In E. coli, Hfq can mediate the interaction between some sRNAs and their target mRNAs. But it is controversial whether Hfq plays an important role in S. aureus. In this study, we found that the deletion of hfq gene in S. aureus 8325-4 can increase the surface carotenoid pigments. The hfq mutant was more resistant to oxidative stress but the pathogenicity of the mutant was reduced. We reveal that the Hfq protein can be detected only in some S. aureus strains. Using microarray and qRT-PCR, we identified 116 genes in the hfq mutant which had differential expression from the wild type, most of which are related to the phenotype and virulence of S. aureus. Among the 116 genes, 49 mRNAs can specifically bind Hfq protein, which indicates that Hfq also acts as an RNA binding protein in S. aureus. Our data suggest that Hfq protein of S. aureus is a multifunctional regulator involved in stress and virulence.

Identification and function of the RNA chaperone Hfq in the Lyme disease spirochete Borrelia burgdorferi.

Hfq is a global regulatory RNA-binding protein. We have identified and characterized an atypical Hfq required for gene regulation and infectivity in the Lyme disease spirochete Borrelia burgdorferi. Sequence analyses of the putative B. burgdorferi Hfq protein revealed only a modest level of similarity with the Hfq from Escherichia coli, although a few key residues are retained and the predicted tertiary structure is similar. Several lines of evidence suggest that the B. burgdorferi bb0268 gene encodes a functional Hfq homologue. First, the hfq(Bb) gene (bb0268) restores the efficient translation of an rpoS::lacZ fusion in an E. coli hfq null mutant. Second, the Hfq from B. burgdorferi binds to the small RNA DsrA(Bb) and the rpoS mRNA. Third, a B. burgdorferi hfq null mutant was generated and has a pleiotropic phenotype that includes increased cell length and decreased growth rate, as found in hfq mutants in other bacteria. The hfq(Bb) mutant phenotype is complemented in trans with the hfq gene from either B. burgdorferi or, surprisingly, E. coli. This is the first example of a heterologous bacterial gene complementing a B. burgdorferi mutant. The alternative sigma factor RpoS and the outer membrane lipoprotein OspC, which are induced by increased temperature and required for mammalian infection, are not upregulated in the hfq mutant. Consequently, the hfq mutant is not infectious by needle inoculation in the murine model. These data suggest that Hfq plays a key role in the regulation of pathogenicity factors in B. burgdorferi and we hypothesize that the spirochete has a complex Hfq-dependent sRNA network.