Broadly conserved FlgV controls flagellar assembly and Borrelia burgdorferi dissemination in mice.

Flagella propel pathogens through their environments yet are expensive to synthesize and are immunogenic. Thus, complex hierarchical regulatory networks control flagellar gene expression. Spirochetes are highly motile bacteria, but peculiarly in the Lyme spirochete Borrelia burgdorferi, the archetypal flagellar regulator σ28 is absent. We rediscovered gene bb0268 in B. burgdorferi as flgV, a broadly-conserved gene in the flagellar superoperon alongside σ28 in many Spirochaetes, Firmicutes and other phyla, with distant homologs in Epsilonproteobacteria. We found that B. burgdorferi FlgV is localized within flagellar motors. B. burgdorferi lacking flgV construct fewer and shorter flagellar filaments and are defective in cell division and motility. During the enzootic cycle, B. burgdorferi lacking flgV survive and replicate in Ixodes ticks but are attenuated for dissemination and infection in mice. Our work defines infection timepoints when spirochete motility is most crucial and implicates FlgV as a broadly distributed structural flagellar component that modulates flagellar assembly.

Extensive diversity in RNA termination and regulation revealed by transcriptome mapping for the Lyme pathogen Borrelia burgdorferi.

Transcription termination is an essential and dynamic process that can tune gene expression in response to diverse molecular signals. Yet, the genomic positions, molecular mechanisms, and regulatory consequences of termination have only been studied thoroughly in model bacteria. Here, we use several RNA-seq approaches to map RNA ends for the transcriptome of the spirochete Borrelia burgdorferi - the etiological agent of Lyme disease. We identify complex gene arrangements and operons, untranslated regions and small RNAs. We predict intrinsic terminators and experimentally test examples of Rho-dependent transcription termination. Remarkably, 63% of RNA 3' ends map upstream of or internal to open reading frames (ORFs), including genes involved in the unique infectious cycle of B. burgdorferi. We suggest these RNAs result from premature termination, processing and regulatory events such as cis-acting regulation. Furthermore, the polyamine spermidine globally influences the generation of truncated mRNAs. Collectively, our findings provide insights into transcription termination and uncover an abundance of potential RNA regulators in B. burgdorferi.

Extensive diversity in RNA termination and regulation revealed by transcriptome mapping for the Lyme pathogen B. burgdorferi.

Transcription termination is an essential and dynamic process that can tune gene expression in response to diverse molecular signals. Yet, the genomic positions, molecular mechanisms, and regulatory consequences of termination have only been studied thoroughly in model bacteria. We employed complementary RNA-seq approaches to map RNA ends for the transcriptome of the spirochete Borrelia burgdorferi - the etiological agent of Lyme disease. By systematically mapping B. burgdorferi RNA ends at single nucleotide resolution, we delineated complex gene arrangements and operons and mapped untranslated regions (UTRs) and small RNAs (sRNAs). We experimentally tested modes of B. burgdorferi transcription termination and compared our findings to observations in E. coli , P. aeruginosa , and B. subtilis . We discovered 63% of B. burgdorferi RNA 3' ends map upstream or internal to open reading frames (ORFs), suggesting novel mechanisms of regulation. Northern analysis confirmed the presence of stable 5' derived RNAs from mRNAs encoding gene products involved in the unique infectious cycle of B. burgdorferi . We suggest these RNAs resulted from premature termination and regulatory events, including forms of cis- acting regulation. For example, we documented that the polyamine spermidine globally influences the generation of truncated mRNAs. In one case, we showed that high spermidine concentrations increased levels of RNA fragments derived from an mRNA encoding a spermidine import system, with a concomitant decrease in levels of the full- length mRNA. Collectively, our findings revealed new insight into transcription termination and uncovered an abundance of potential RNA regulators.

BB0562 is a nutritional virulence determinant with lipase activity important for Borrelia burgdorferi infection and survival in fatty acid deficient environments.

The Lyme disease spirochete Borrelia burgdorferi relies on uptake of essential nutrients from its host environments for survival and infection. Therefore, nutrient acquisition mechanisms constitute key virulence properties of the pathogen, yet these mechanisms remain largely unknown. In vivo expression technology applied to B. burgdorferi (BbIVET) during mammalian infection identified gene bb0562, which encodes a hypothetical protein comprised of a conserved domain of unknown function, DUF3996. DUF3996 is also found across adjacent encoded hypothetical proteins BB0563 and BB0564, suggesting the possibility that the three proteins could be functionally related. Deletion of bb0562, bb0563 and bb0564 individually and together demonstrated that bb0562 alone was important for optimal disseminated infection in immunocompetent and immunocompromised mice by needle inoculation and tick bite transmission. Moreover, bb0562 promoted spirochete survival during the blood dissemination phase of infection. Gene bb0562 was also found to be important for spirochete growth in low serum media and the growth defect of Δbb0562 B. burgdorferi was rescued with the addition of various long chain fatty acids, particularly oleic acid. In mammals, fatty acids are primarily stored in fat droplets in the form of triglycerides. Strikingly, addition of glyceryl trioleate, the triglyceride form of oleic acid, to the low serum media did not rescue the growth defect of the mutant, suggesting bb0562 may be important for the release of fatty acids from triglycerides. Therefore, we searched for and identified two canonical GXSXG lipase motifs within BB0562, despite the lack of homology to known bacterial lipases. Purified BB0562 demonstrated lipolytic activity dependent on the catalytic serine residues within the two motifs. In sum, we have established that bb0562 is a novel nutritional virulence determinant, encoding a lipase that contributes to fatty acid scavenge for spirochete survival in environments deficient in free fatty acids including the mammalian host.

Regulatory roles of Escherichia coli 5' UTR and ORF-internal RNAs detected by 3' end mapping.

Many bacterial genes are regulated by RNA elements in their 5´ untranslated regions (UTRs). However, the full complement of these elements is not known even in the model bacterium Escherichia coli. Using complementary RNA-sequencing approaches, we detected large numbers of 3´ ends in 5´ UTRs and open reading frames (ORFs), suggesting extensive regulation by premature transcription termination. We documented regulation for multiple transcripts, including spermidine induction involving Rho and translation of an upstream ORF for an mRNA encoding a spermidine efflux pump. In addition to discovering novel sites of regulation, we detected short, stable RNA fragments derived from 5´ UTRs and sequences internal to ORFs. Characterization of three of these transcripts, including an RNA internal to an essential cell division gene, revealed that they have independent functions as sRNA sponges. Thus, these data uncover an abundance of cis- and trans-acting RNA regulators in bacterial 5´ UTRs and internal to ORFs.

In most organisms, specific segments of a cell's genetic information are copied to form single-stranded molecules of various sizes and purposes. Each of these RNA molecules, as they are known, is constructed as a chain that starts at the 5´ end and terminates at the 3´ end. Certain RNAs carry the information present in a gene, which provides the instructions that a cell needs to build proteins. Some, however, are 'non-coding' and instead act to fine-tune the activity of other RNAs. These regulatory RNAs can be separate from the RNAs they control, or they can be embedded in the very sequences they regulate; new evidence also shows that certain regulatory RNAs can act in both ways. Many regulatory RNAs are yet to be catalogued, even in simple, well-studied species such as the bacterium Escherichia coli. Here, Adams et al. aimed to better characterize the regulatory RNAs present in E. coli by mapping out the 3´ ends of every RNA molecule in the bacterium. This revealed many new regulatory RNAs and offered insights into where these sequences are located. For instance, the results show that several of these RNAs were embedded within RNA produced from larger genes. Some were nested in coding RNAs, and were parts of a longer RNA sequence that is adjacent to the protein coding segment. Others, however, were present within the instructions that code for a protein. The work by Adams et al. reveals that regulatory RNAs can be located in unexpected places, and provides a method for identifying them. This can be applied to other types of bacteria, in particular in species with few known RNA regulators.

Prevalence of small base-pairing RNAs derived from diverse genomic loci.

Small RNAs (sRNAs) that act by base-pairing have been shown to play important roles in fine-tuning the levels and translation of their target transcripts across a variety of model and pathogenic organisms. Work from many different groups in a wide range of bacterial species has provided evidence for the importance and complexity of sRNA regulatory networks, which allow bacteria to quickly respond to changes in their environment. However, despite the expansive literature, much remains to be learned about all aspects of sRNA-mediated regulation, particularly in bacteria beyond the well-characterized Escherichia coli and Salmonella enterica species. Here we discuss what is known, and what remains to be learned, about the identification of regulatory base-pairing RNAs produced from diverse genomic loci including how their expression is regulated. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.

RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles.

Base pairing RNAs modulate gene expression in all studied organisms. In many bacteria, the base pairing between most small regulatory RNAs (sRNAs) and their targets is mediated by the Hfq RNA chaperone. However, recent studies have shown FinO-domain proteins also bind sRNAs. To examine the global contribution of the FinO-domain ProQ protein in Escherichia coli, we carried out RIL-seq to identify RNA pairs bound to this protein. The RNA-RNA interactome for ProQ contains hundreds of pairs. Intriguingly, a significant fraction of the ProQ-bound RNA pairs are also found associated with Hfq, indicating overlapping, complementary, or competing roles for the two proteins. Characterization of one novel RNA pair bound by both chaperones revealed that while Hfq is required for RNA sponge-mediated downregulation of the sRNA, ProQ can inhibit this regulation. Overall, our results uncover increased complexity in RNA regulatory networks involving RNA chaperone proteins, RNases, sRNAs, and mRNAs.

Borrelia burgdorferi bbk13 Is Critical for Spirochete Population Expansion in the Skin during Early Infection.

Lyme disease is caused by the spirochete Borrelia burgdorferi and is transmitted via the bite of an infected tick. B. burgdorferi enters the skin, disseminates via the bloodstream, and infects various distal tissues, leading to inflammatory sequelae, such as Lyme arthritis and Lyme carditis. B. burgdorferi linear plasmid 36 (lp36) is critical for mammalian infectivity; however, the full complement of genes on lp36 that contribute to this process remains unknown. Through a targeted mutagenesis screen of the genes on lp36, we identified a novel infectivity gene of unknown function, bbk13, which encodes an immunogenic, non-surface-exposed membrane protein that is important for efficient mammalian infection. Loss of bbk13 resulted in reduced spirochete loads in distal tissues in a mouse model of infection. Through a detailed analysis of B. burgdorferi infection kinetics, we discovered that bbk13 is important for promoting spirochete proliferation in the skin inoculation site. The attenuated ability of Δbbk13 spirochetes to proliferate in the inoculation site was followed by reduced numbers of B. burgdorferi spirochetes in the bloodstream and, ultimately, consistently reduced spirochete loads in distal tissues. Together, our data indicate that bbk13 contributes to disseminated infection by promoting spirochete proliferation in the early phase of infection in the skin. This work not only increases the understanding of the contribution of the genes on lp36 to B. burgdorferi infection but also begins to define the genetic basis for B. burgdorferi expansion in the skin during localized infection and highlights the influence of the early expansion of spirochetes in the skin on the outcome of infection.

Selection of Borrelia burgdorferi Promoter Sequences Active During Mammalian Infection Using In Vivo Expression Technology.

In vivo expression technology (IVET) has been applied to a variety of organisms to identify active promoters in specific environments or growth conditions of interest. Here, we describe modifications to employ this genome-wide screening method for Borrelia burgdorferi, the Lyme disease spirochete, during an active murine infection. Utilization of this technique provides valuable insights into the B. burgdorferi transcriptome during infection, despite the low bacterial numbers in the mammalian host environment.

A Dual Luciferase Reporter System for B. burgdorferi Measures Transcriptional Activity during Tick-Pathogen Interactions.

Knowledge of the transcriptional responses of vector-borne pathogens at the vector-pathogen interface is critical for understanding disease transmission. Borrelia (Borreliella) burgdorferi, the causative agent of Lyme disease in the United States, is transmitted by the bite of infected Ixodes sp. ticks. It is known that B. burgdorferi has altered patterns of gene expression during tick acquisition, persistence and transmission. Recently, we and others have discovered in vitro expression of RNAs found internal, overlapping, and antisense to annotated open reading frames in the B. burgdorferi genome. However, there is a lack of molecular genetic tools for B. burgdorferi for quantitative, strand-specific, comparative analysis of these transcripts in distinct environments such as the arthropod vector. To address this need, we have developed a dual luciferase reporter system to quantify B. burgdorferi promoter activities in a strand-specific manner. We demonstrate that constitutive expression of a B. burgdorferi codon-optimized Renilla reniformis luciferase gene (rlucBb ) allows normalization of the activity of a promoter of interest when fused to the B. burgdorferi codon-optimized Photinus pyralis luciferase gene (flucBb) on the same plasmid. Using the well characterized, differentially regulated, promoters for flagellin (flaBp), outer surface protein A (ospAp) and outer surface protein C (ospCp), we document the efficacy of the dual luciferase system for quantitation of promoter activities during in vitro growth and in infected ticks. Cumulatively, the dual luciferase method outlined herein is the first dual reporter system for B. burgdorferi, providing a novel and highly versatile approach for strand-specific molecular genetic analyses.

In vivo expression technology and 5' end mapping of the Borrelia burgdorferi transcriptome identify novel RNAs expressed during mammalian infection.

Borrelia burgdorferi, the bacterial pathogen responsible for Lyme disease, modulates its gene expression profile in response to the environments encountered throughout its tick-mammal infectious cycle. To begin to characterize the B. burgdorferi transcriptome during murine infection, we previously employed an in vivo expression technology-based approach (BbIVET). This identified 233 putative promoters, many of which mapped to un-annotated regions of the complex, segmented genome. Herein, we globally identify the 5' end transcriptome of B. burgdorferi grown in culture as a means to validate non-ORF associated promoters discovered through BbIVET. We demonstrate that 119 BbIVET promoters are associated with transcription start sites (TSSs) and validate novel RNA transcripts using Northern blots and luciferase promoter fusions. Strikingly, 49% of BbIVET promoters were not found to associate with TSSs. This finding suggests that these sequences may be primarily active in the mammalian host. Furthermore, characterization of the 6042 B. burgdorferi TSSs reveals a variety of RNAs including numerous antisense and intragenic transcripts, leaderless RNAs, long untranslated regions and a unique nucleotide frequency for initiating intragenic transcription. Collectively, this is the first comprehensive map of TSSs in B. burgdorferi and characterization of previously un-annotated RNA transcripts expressed by the spirochete during murine infection.

Gene bb0318 Is Critical for the Oxidative Stress Response and Infectivity of Borrelia burgdorferi.

A greater understanding of the molecular mechanisms that Borrelia burgdorferi uses to survive during mammalian infection is critical for the development of novel diagnostic and therapeutic tools to improve the clinical management of Lyme disease. By use of an in vivo expression technology (IVET)-based approach to identify B. burgdorferi genes expressed in vivo, we discovered the bb0318 gene, which is thought to encode the ATPase component of a putative riboflavin ABC transport system. Riboflavin is a critical metabolite enabling all organisms to maintain redox homeostasis. B. burgdorferi appears to lack the metabolic capacity for de novo synthesis of riboflavin and so likely relies on scavenging riboflavin from the host environment. In this study, we sought to investigate the role of bb0318 in B. burgdorferi pathogenesis. No in vitro growth defect was observed for the Δbb0318 clone. However, the mutant spirochetes displayed reduced levels of survival when exposed to exogenous hydrogen peroxide or murine macrophages. Spirochetes lacking bb0318 were found to have a 100-fold-higher 50% infectious dose than spirochetes containing bb0318 In addition, at a high inoculum dose, bb0318 was found to be important for effective spirochete dissemination to deep tissues for as long as 3 weeks postinoculation and to be critical for B. burgdorferi infection of mouse hearts. Together, these data implicate bb0318 in the oxidative stress response of B. burgdorferi and indicate the contribution of bb0318 to B. burgdorferi mammalian infectivity.