Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein.

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G-C content play a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen.

The σ54-σS sigma factor cascade plays a central role in regulating differential gene expression during the enzootic cycle of Borreliella burgdorferi, the Lyme disease pathogen. In this pathway, the primary transcription of rpoS (which encodes σS) is under the control of σ54 which is activated by a bacterial enhancer-binding protein (EBP), Rrp2. The σ54-dependent activation in B. burgdorferi has long been thought to be unique, requiring an additional factor, BosR, a homologue of classical Fur/PerR repressor/activator. However, how BosR is involved in this σ54-dependent activation remains unclear and perplexing. In this study, we demonstrate that BosR does not function as a regulator for rpoS transcriptional activation. Instead, it functions as a novel RNA-binding protein that governs the turnover rate of rpoS mRNA. We further show that BosR directly binds to the 5' untranslated region (UTR) of rpoS mRNA, and the binding region overlaps with a region required for rpoS mRNA degradation. Mutations within this 5'UTR region result in BosR-independent RpoS production. Collectively, these results uncover a novel role of Fur/PerR family regulators as RNA-binding proteins and redefine the paradigm of the σ54-σS pathway in B. burgdorferi.

Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein.

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length plays a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

Quantitative analyses of interactions between SpoVG and RNA/DNA.

The Borrelia burgdorferi SpoVG protein has previously been found to be a DNA- and RNA-binding protein. To aid in the elucidation of ligand motifs, affinities for numerous RNAs, ssDNAs, and dsDNAs were measured and compared. The loci used in the study were spoVG, glpFKD, erpAB, bb0242, flaB, and ospAB, with particular focus on the untranslated 5' portion of the mRNAs. Performing binding and competition assays yielded that the 5' end of spoVG mRNA had the highest affinity while the lowest observed affinity was to the 5' end of flaB mRNA. Mutagenesis studies of spoVG RNA and ssDNA sequences suggested that the formation of SpoVG-nucleic acid complexes are not entirely dependent on either sequence or structure. Additionally, exchanging uracil for thymine in ssDNAs did not affect protein-nucleic acid complex formation.

Quantitative analyses of interactions between SpoVG and RNA/DNA.

The Borrelia burgdorferi SpoVG protein has previously been found to be a DNA- and RNA-binding protein. To aid in the elucidation of ligand motifs, affinities for numerous RNAs, ssDNAs, and dsDNAs were measured and compared. The loci used in the study were spoVG, glpFKD, erpAB, bb0242, flaB, and ospAB, with particular focus on the untranslated 5' portion of the mRNAs. Performing binding and competition assays yielded that the 5' end of spoVG mRNA had the highest affinity while the lowest observed affinity was to the 5' end of flaB mRNA. Mutagenesis studies of spoVG RNA and ssDNA sequences suggested that the formation of SpoVG-nucleic acid complexes are not entirely dependent on either sequence or structure. Additionally, exchanging uracil for thymine in ssDNAs did not affect protein-nucleic acid complex formation.

Transcript decay mediated by RNase III in Borrelia burgdorferi.

The causative agent of Lyme disease, Borrelia burgdorferi, requires shifts in gene expression to undergo its natural enzootic cycle between tick and vertebrate hosts. mRNA decay mechanisms play significant roles in governing gene expression in other bacteria, but are not yet characterized in B. burgdorferi. RNase III is an important enzyme in processing ribosomal RNA, but it also plays a role in mRNA decay in many bacteria. We compared RNA decay profiles and steady-state abundances of transcripts in wild-type Borrelia burgdorferi strain B31 and in an RNase III null (rnc-) mutant. Transcripts encoding RNA polymerase subunits (rpoA and rpoS), ribosomal proteins (rpsD, rpsK, rpsM, rplQ, and rpsO), a nuclease (pnp), a flagellar protein (flaB), and a translational regulator (bpuR) decayed more rapidly in the wild-type strain than in the slow growing rnc- mutant indicating that RNA turnover is mediated by RNase III in the bacterium that causes Lyme disease. Additionally, in wild type bacteria, RNA decay rates of rpoS, rpoN, ospA, ospC, bpuR and dbpA transcripts are only modestly affected by changes in the osmolarity.

Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans.

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (ß-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the ß-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of ß-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.

Measurements of mRNA degradation in Borrelia burgdorferi.

The importance of gene regulation in the enzootic cycle of Borrelia burgdorferi, the spirochete that causes Lyme disease, is well established. B. burgdorferi regulates gene expression in response to changes in environmental stimuli associated with changing hosts. In this study, we monitored mRNA decay in B. burgdorferi following transcriptional arrest with actinomycin D. The time-dependent decay of transcripts encoding RNA polymerase subunits (rpoA and rpoS), ribosomal proteins (rpsD, rpsK, rpsM, rplQ, and rpsO), a nuclease (pnp), outer surface lipoproteins (ospA and ospC), and a flagellar protein (flaB) have different profiles and indicate half-lives ranging from approximately 1 min to more than 45 min in cells cultured at 35°C. Our results provide a first step in characterizing mRNA decay in B. burgdorferi and in investigating its role in gene expression and regulation.

Translational efficiency of rpoS mRNA from Borrelia burgdorferi: effects of the length and sequence of the mRNA leader region.

Regulation of the enzootic cycle in Borrelia burgdorferi requires a shift to the RNA polymerase alternative sigma factor, RpoS. We used in vitro and in vivo assays to assess the relative importance of the putative Shine-Dalgarno sequence and its sequestration for the translational efficiency of rpoS. We created mutant leader regions in which we either removed the Shine-Dalgarno sequence, disrupted the secondary structure or both. Binding assays and toeprint assays demonstrated that both the presence and the availability of the Shine-Dalgarno sequence are important to the efficiency and specificity of ribosome binding. Adding a DsrABb mimic in the form of a single-stranded DNA oligonucleotide increased the level and specificity of binding ribosomes to the transcript with an extended leader, presumably by making the Shine-Dalgarno sequence available for binding. In in vivo assays we confirmed that the Shine-Dalgarno sequence must be both present and un-sequestered in order for translation to proceed efficiently. The longer transcript was significantly better translated in B. burgdorferi at 37 °C than at 26 °C, lending support to the hypothesis that DsrABb acts as a temperature-dependent stimulator of translation. These studies demonstrate that translational regulation of gene expression in B. burgdorferi may be an important mechanism for responding to environmental signals important in the enzootic cycle.

Interactions of ribosomal protein S1 with DsrA and rpoS mRNA.

Ribosomal protein S1 is shown to interact with the non-coding RNA DsrA and with rpoS mRNA. DsrA is a non-coding RNA that is important in controlling expression of the rpoS gene product in Escherichia coli. Photochemical crosslinking, quadrupole-time of flight tandem mass spectrometry, and peptide sequencing have identified an interaction between DsrA and S1 in the 30S ribosomal subunit. Purified S1 binds both DsrA (K(obs) approximately 6 x 10(6) M(-1)) and rpoS mRNA (K(obs) approximately 3 x 10(7) M(-1)). Ribonuclease probing experiments indicate that S1 binding has a weak but detectable effect on the secondary structure of DsrA or rpoS mRNA.

Translational repression mechanisms in prokaryotes.

Translational repression results from a complex choreography of macromolecular interactions interfering with the formation of translational initiation complexes. The relationship between the rate and extent of formation of these interactions to form repressed mRNA complexes determines the extent of repression. A novel analysis of repression mechanisms is presented here and it indicates that the reversibility of repressed complex formation influences the steady state balance of the distribution of translationally active and inactive complexes and therefore has an impact on the efficiency of repression. Reviewed here is evidence for three distinct translational repression mechanisms, regulating expression of the transcription factor sigma32, threonine tRNA synthetase and ribosomal proteins on the alpha operon in Escherichia coli. Efficient regulation of expression in these systems makes use of specific mRNA structures in quite different ways.

Interactions of the non-coding RNA DsrA and RpoS mRNA with the 30 S ribosomal subunit.

Expression of sigma(s), the gene product of rpoS, is controlled translationally in response to many environmental stresses. DsrA, a small 87-nucleotide non-coding RNA molecule, acts to increase translational efficiency of RpoS mRNA under some growth conditions. In this work, we demonstrate that DsrA binds directly to the 30 S ribosomal subunit with an observed equilibrium affinity of 2.8 x 10(7) m(-1). DsrA does not compete with RpoS mRNA or tRNA(f)(Met) for binding to the 30 S subunit. The 5' end of DsrA binds to 30 S subunits with an observed equilibrium association constant of 2.0 x 10(6) m(-1), indicating that the full affinity of the interaction requires the entire DsrA sequence. In order to investigate translational efficiency of RpoS mRNA, we examined both ribosome-binding site accessibility and the binding of RpoS mRNA to 30 S ribosomal subunits. We find that that ribosome-binding site accessibility is modulated as a function of divalent cation concentration during mRNA renaturation and by the presence of an antisense sequence that binds to nucleotides 1-16 of the RpoS mRNA fragment. The ribosome-binding site accessibility correlates with the amount of RpoS mRNA participating in 30 S-mRNA "pre-initiation" translational complex formation and provides evidence that regulation follows a competitive model of regulation.