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.

c-di-GMP regulates activity of the PlzA RNA chaperone from the Lyme disease spirochete.

PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for survival in the tick; however, the biological function of PlzA has remained enigmatic. Here, we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding. Holo- and apo-PlzA bind RNA and accelerate RNA annealing, while only apo-PlzA can strand displace and unwind double-stranded RNA. Guided by the crystal structure of PlzA, we identified several key aromatic amino acids protruding from the N- and C-terminal domains that are required for RNA-binding and unwinding activity. Our findings illuminate c-di-GMP as a switch controlling the RNA chaperone activity of PlzA, and we propose that complex RNA-mediated modulatory mechanisms allow PlzA to regulate gene expression during both the vector and host phases of the B. burgdorferi life cycle.

The glycerol-3-phosphate dehydrogenases GpsA and GlpD constitute the oxidoreductive metabolic linchpin for Lyme disease spirochete host infectivity and persistence in the tick.

We have identified GpsA, a predicted glycerol-3-phosphate dehydrogenase, as a virulence factor in the Lyme disease spirochete Borrelia (Borreliella) burgdorferi: GpsA is essential for murine infection and crucial for persistence of the spirochete in the tick. B. burgdorferi has a limited biosynthetic and metabolic capacity; the linchpin connecting central carbohydrate and lipid metabolism is at the interconversion of glycerol-3-phosphate and dihydroxyacetone phosphate, catalyzed by GpsA and another glycerol-3-phosphate dehydrogenase, GlpD. Using a broad metabolomics approach, we found that GpsA serves as a dominant regulator of NADH and glycerol-3-phosphate levels in vitro, metabolic intermediates that reflect the cellular redox potential and serve as a precursor for lipid and lipoprotein biosynthesis, respectively. Additionally, GpsA was required for survival under nutrient stress, regulated overall reductase activity and controlled B. burgdorferi morphology in vitro. Furthermore, during in vitro nutrient stress, both glycerol and N-acetylglucosamine were bactericidal to B. burgdorferi in a GlpD-dependent manner. This study is also the first to identify a suppressor mutation in B. burgdorferi: a glpD deletion restored the wild-type phenotype to the pleiotropic gpsA mutant, including murine infectivity by needle inoculation at high doses, survival under nutrient stress, morphological changes and the metabolic imbalance of NADH and glycerol-3-phosphate. These results illustrate how basic metabolic functions that are dispensable for in vitro growth can be essential for in vivo infectivity of B. burgdorferi and may serve as attractive therapeutic targets.

An Antisense RNA Fine-Tunes Gene Expression of the Type II MazEF Toxin-Antitoxin System.

Despite their ubiquitous nature, few antisense RNAs have been functionally characterized, and this class of RNAs is considered by some to be transcriptional noise. Here, we report that an antisense RNA (asRNA), aMEF (antisense mazEF), functions as a dual regulator for the type II toxin-antitoxin (TA) system mazEF. Unlike type I TA systems and many other regulatory asRNAs, aMEF stimulates the synthesis and translation of mazEF rather than inhibition and degradation. Our data indicate that a double-stranded RNA intermediate and RNase III are not necessary for aMEF-dependent regulation of mazEF expression. The lack of conservation of asRNA promoters has been used to support the hypothesis that asRNAs are spurious transcriptional noise and nonfunctional. We demonstrate that the aMEF promoter is active and functional in Escherichia coli despite poor sequence conservation, indicating that the lack of promoter sequence conservation should not be correlated with functionality. IMPORTANCE Next-generation RNA sequencing of numerous organisms has revealed that transcription is widespread across the genome, termed pervasive transcription, and does not adhere to annotated gene boundaries. The function of pervasive transcription is enigmatic and has generated considerable controversy as to whether it is transcriptional noise or biologically relevant. Antisense transcription is one class of pervasive transcription that occurs from the DNA strand opposite an annotated gene. Relatively few pervasively transcribed asRNAs have been functionally characterized, and their regulatory roles or lack thereof remains unknown. It is important to study examples of these asRNAs and determine if they are functional regulators. In this study, we elucidate the function of an asRNA (aMEF) demonstrating that pervasive transcripts can be functional.

The Borrelia burgdorferi Adenylate Cyclase, CyaB, Is Important for Virulence Factor Production and Mammalian Infection.

Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3' end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full-length SR0623 to evaluate the influence on transcriptional and posttranscriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, the expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.

Gene Regulation and Transcriptomics.

Borrelia (Borreliella) burgdorferi, along with closely related species, is the etiologic agent of Lyme disease. The spirochete subsists in an enzootic cycle that encompasses acquisition from a vertebrate host to a tick vector and transmission from a tick vector to a vertebrate host. To adapt to its environment and persist in each phase of its enzootic cycle, B. burgdorferi wields three systems to regulate the expression of genes: the RpoN-RpoS alternative sigma factor cascade, the Hk1/Rrp1 two-component system and its product c-di-GMP, and the stringent response mediated by RelBbu and DksA. These regulatory systems respond to enzootic phase-specific signals and are controlled or fine- tuned by transcription factors, including BosR and BadR, as well as small RNAs, including DsrABb and Bb6S RNA. In addition, several other DNA-binding and RNA-binding proteins have been identified, although their functions have not all been defined. Global changes in gene expression revealed by high-throughput transcriptomic studies have elucidated various regulons, albeit technical obstacles have mostly limited this experimental approach to cultivated spirochetes. Regardless, we know that the spirochete, which carries a relatively small genome, regulates the expression of a considerable number of genes required for the transitions between the tick vector and the vertebrate host as well as the adaptation to each.

Shep interacts with posttranscriptional regulators to control dendrite morphogenesis in sensory neurons.

RNA binding proteins (RBPs) mediate posttranscriptional gene regulatory events throughout development. During neurogenesis, many RBPs are required for proper dendrite morphogenesis within Drosophila sensory neurons. Despite their fundamental role in neuronal morphogenesis, little is known about the molecular mechanisms in which most RBPs participate during neurogenesis. In Drosophila, alan shepard (shep) encodes a highly conserved RBP that regulates dendrite morphogenesis in sensory neurons. Moreover, the C. elegans ortholog sup-26 has also been implicated in sensory neuron dendrite morphogenesis. Nonetheless, the molecular mechanism by which Shep/SUP-26 regulate dendrite development is not understood. Here we show that Shep interacts with the RBPs Trailer Hitch (Tral), Ypsilon schachtel (Yps), Belle (Bel), and Poly(A)-Binding Protein (PABP), to direct dendrite morphogenesis in Drosophila sensory neurons. Moreover, we identify a conserved set of Shep/SUP-26 target RNAs that include regulators of cell signaling, posttranscriptional gene regulators, and known regulators of dendrite development.

Small RNAs of Borrelia burgdorferi: Characterizing Functional Regulators in a Sea of sRNAs
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Borrelia (Borreliella) burgdorferi and closely related genospecies are the causative agents of Lyme disease, the most common tick-borne disease north of the equator. The bacterium, a member of the spirochete phylum, is acquired by a tick vector that feeds on an infected vertebrate host and is transmitted to another vertebrate during subsequent feeding by the next tick stage. The precise navigation of this enzootic cycle entails the regulation of genes required for these two host-specific phases as well as the transitions between them. Recently, an expansive swath of small RNAs has been identified in B. burgdorferi and likely many, if not most, are involved in regulating gene expression. Regardless, with only a few exceptions, the functions of these RNAs are completely unknown. However, several state-of-the-art approaches are available to identify the targets of these RNAs and provide insight into their role in the enzootic cycle and infection.

The RNA-binding protein caper is required for sensory neuron development in Drosophila melanogaster.

BACKGROUND: Alternative splicing mediated by RNA-binding proteins (RBPs) is emerging as a fundamental mechanism for the regulation of gene expression. Alternative splicing has been shown to be a widespread phenomenon that facilitates the diversification of gene products in a tissue-specific manner. Although defects in alternative splicing are rooted in many neurological disorders, only a small fraction of splicing factors have been investigated in detail. RESULTS: We find that the splicing factor Caper is required for the development of multiple different mechanosensory neuron subtypes at multiple life stages in Drosophila melanogaster. Disruption of Caper function causes defects in dendrite morphogenesis of larval dendrite arborization neurons and neuronal positioning of embryonic proprioceptors, as well as the development and maintenance of adult mechanosensory bristles. Additionally, we find that Caper dysfunction results in aberrant locomotor behavior in adult flies. Transcriptome-wide analyses further support a role for Caper in alternative isoform regulation of genes that function in neurogenesis. CONCLUSIONS: Our results provide the first evidence for a fundamental and broad requirement for the highly conserved splicing factor Caper in the development and maintenance of the nervous system and provide a framework for future studies on the detailed mechanism of Caper-mediated RNA regulation. Developmental Dynamics 246:610-624, 2017. © 2017 Wiley Periodicals, Inc.

Borrelia burgdorferi malQ mutants utilize disaccharides and traverse the enzootic cycle.

Borrelia burgdorferi, the causative agent of Lyme disease, cycles in nature between a vertebrate host and a tick vector. We demonstrate that B. burgdorferi can utilize several sugars that may be available during persistence in the tick, including trehalose, N-acetylglucosamine (GlcNAc), and chitobiose. The spirochete grows to a higher cell density in trehalose, which is found in tick hemolymph, than in maltose; these two disaccharides differ only in the glycosidic linkage between the glucose monomers. Additionally, B. burgdorferi grows to a higher density in GlcNAc than in the GlcNAc dimer chitobiose, both of which may be available during tick molting. We have also investigated the role of malQ (bb0166), which encodes an amylomaltase, in sugar utilization during the enzootic cycle. In other bacteria, MalQ is involved in utilizing maltodextrins and trehalose, but we show that, unexpectedly, it is not needed for B. burgdorferi to grow in vitro on any of the sugars assayed. In addition, infection of mice by needle inoculation or tick bite, as well as acquisition and maintenance of the spirochete in the tick vector, does not require MalQ.

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.

Temperature-induced regulation of RpoS by a small RNA in Borrelia burgdorferi.

The alternative sigma factor RpoS (sigma38 or sigmaS) plays a central role in the reciprocal regulation of the virulence-associated major outer surface proteins OspC and OspA in Borrelia burgdorferi, the Lyme disease spirochete. Temperature is one of the key environmental signals controlling RpoS, but the molecular mechanism by which the signal is transduced remains unknown. Herein, we identify and describe a small non-coding RNA, DsrABb, that regulates the temperature-induced increase in RpoS. A novel 5' end of the rpoS mRNA was identified and DsrABb has the potential to extensively base-pair with the upstream region of this rpoS transcript. We demonstrate that B. burgdorferi strains lacking DsrABb do not upregulate RpoS and OspC in response to an increase in temperature, but do regulate RpoS and OspC in response to changes in pH and cell density. Analyses of the rpoS and ospC steady-state mRNA levels in the dsrABb mutant indicate that DsrABb regulates RpoS post-transcriptionally. The 5' and 3' ends of DsrABb were mapped, demonstrating that at least four species exist with sizes ranging from 213 to 352 nucleotides. We hypothesize that DsrABb binds to the upstream region of the rpoS mRNA and stimulates translation by releasing the Shine-Dalgarno sequence and start site from a stable secondary structure. Therefore, we postulate that DsrABb is a molecular thermometer regulating RpoS in Borrelia burgdorferi.

Analysis of the ospC regulatory element controlled by the RpoN-RpoS regulatory pathway in Borrelia burgdorferi.

Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi. ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS (sigma(s)) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical -35/-10 sequence necessary and sufficient for sigma(s)-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position -15 within the extended -10 region of ospC, which is postulated to function like the strategic C residue important for Esigma(s) binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin A(1), further supporting its sigma(s) character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of sigma(s) to a sigma(s)-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.

Transcriptional regulation of the ospAB and ospC promoters from Borrelia burgdorferi.

OspA, OspB and OspC are the major outer surface proteins of Borrelia burgdorferi that are differentially synthesized in response to environmental conditions, including culture temperature. We found that DNA was more negatively supercoiled in B. burgdorferi cultures grown at 23 degrees C compared with cultures grown at 35-37 degrees C. We examined the regulation of ospAB and ospC transcription by temperature and DNA supercoiling. DNA supercoiling was relaxed by adding coumermycin A1, an antibiotic that inhibits DNA gyrase. Syntheses of the major outer surface proteins, expression of the ospA and ospC genes and the activities of the ospAB operon and ospC gene promoters were assayed. ospA product levels decreased, whereas ospC product levels increased after shifting from 23 degrees C to 35 degrees C or after adding coumermycin A1. In addition, OspC synthesis was higher in a gyrB mutant than in wild-type B. burgdorferi. Promoter activity was quantified using cat reporter fusions. Increasing temperature or relaxing supercoiled DNA resulted in a decrease in ospAB promoter activity in B. burgdorferi, but not in Escherichia coli, as well as an increase in ospC promoter activity in both bacteria. ospC promoter activity was increased in an E. coli gyrB mutant with an attenuated DNA supercoiling phenotype. These results suggest that B. burgdorferi senses environmental changes in temperature by altering the level of DNA supercoiling, which then affects the expression of the ospAB operon and the ospC gene. This implies that DNA supercoiling acts as a signal transducer for environmental regulation of outer surface protein synthesis.