Efficient and highly organized regulation of transcription is fundamental to an organism’s ability to survive, proliferate, and quickly respond to its environment. Therefore, precise mapping of transcriptional units and understanding their regulation is crucial to determining how pathogenic bacteria cause disease and how they may be inhibited. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae TIGR4 by applying a combination of high-throughput RNA-sequencing techniques. We successfully map 1864 high confidence transcription termination sites (TTSs), 790 high confidence transcription start sites (TSSs) (742 primary, and 48 secondary), and 1360 low confidence TSSs (74 secondary and 1286 primary) to yield a total of 2150 TSSs. Furthermore, our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal TSSs and TTSs. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5’-untranslated regions (5’-UTR). By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to demonstrate the importance of ribo-regulation by 5’-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-)regulators to mitigate virulence and antibiotic resistance.

Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated (≥2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 5′ UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during transcription.

The intervening sequence (IVS) of Coxiella burnetii, the agent of Q fever, is a 428-nt selfish genetic element located in helix 45 of the precursor 23S rRNA. The IVS element, in turn, contains an ORF that encodes a hypothetical ribosomal S23 protein (S23p). Although S23p can be synthesized in vitro in the presence of an engineered E. coli promoter and ribosome binding site, results suggest that the protein is not synthesized in vivo. In spite of a high degree of IVS conservation among different strains of C. burnetii, the region immediately upstream of the S23p start codon is prone to change, and the S23p-encoding ORF is evidently undergoing reductive evolution. We determined that IVS excision from 23S rRNA was mediated by RNase III, and IVS RNA was rapidly degraded, thereafter. Levels of the resulting 23S rRNA fragments that flank the IVS, F1 (~1.2 kb) and F2 (~1.7 kb), were quantified over C. burnetii's logarithmic growth phase (1–5 d). Results showed that 23S F1 quantities were consistently higher than those of F2 and 16S rRNA. The disparity between levels of the two 23S rRNA fragments following excision of IVS is an interesting phenomenon of unknown significance. Based upon phylogenetic analyses, IVS was acquired through horizontal transfer after C. burnetii's divergence from an ancestral bacterium and has been subsequently maintained by vertical transfer. The widespread occurrence, maintenance and conservation of the IVS in C. burnetii imply that it plays an adaptive role or has a neutral effect on fitness.

The 23S rRNA gene of Coxiella burnetii, the agent of Q fever in humans, contains an unusually high number of conserved, selfish genetic elements, including two group I introns, termed Cbu.L1917 (L1917) and Cbu.L1951 (L1951). To better understand the role that introns play in Coxiella's biology, we determined the intrinsic stability time periods (in vitro half-lives) of the encoded ribozymes to be ϳ15 days for L1917 and ϳ5 days for L1951, possibly due to differences in their sizes (551 and 1,559 bases, respectively), relative degrees of compactness of the respective RNA structures, and amounts of single-stranded RNA. In vivo half-lives for both introns were also determined to be ϳ11 min by the use of RNase protection assays and an Escherichia coli model. Intron RNAs were quantified in synchronous cultures of C. burnetii and found to closely parallel those of 16S rRNA; i.e., ribozyme levels significantly increased between days 0 and 3 and then remained stable until 8 days postinfection. Both 16S rRNA and ribozyme levels fell during the stationary and death phases (days 8 to 14). The marked stability of the Coxiella intron RNAs is presumably conferred by their association with ribosomes, a stoichiometric relationship that was determined to be one ribozyme, of either type, per 500 ribosomes. Inaccuracies in splicing (exon 2 skipping) were found to increase during the first 5 days in culture, with a rate of approximately one improperly spliced 23S rRNA per 1.3 million copies. The in vitro efficiency of L1917 intron splicing was significantly enhanced in the presence of a recombinant Coxiella RNA DEAD-box helicase (CBU_0670) relative to that of controls, suggesting that this enzyme may serve as an intron RNA splice facilitator in vivo.

32Efficient and highly organized transcription initiation and termination is fundamental to an 33 organism's ability to survive, proliferate, and quickly respond to its environment. Over the last 34 decade, our simplistic outlook of bacterial transcriptional regulation and architecture has evolved 35 to include stimulus-responsive regulation by untranslated RNA and the formation of alternate 36 transcriptional units. In this study, we map the transcriptional landscape of the bacterial pathogen 37 Streptococcus pneumoniae by applying a combination of high-throughput RNA-sequencing 38 techniques. Our study reveals a complex transcriptome wherein environment-respondent 39 alternate transcriptional units are observed within operons stemming from internal transcription 40 start sites (TSS) and transcription terminators (TTS) suggesting that more fine-tuning of 41 regulation occurs than previously thought. Additionally, we identify many putative cis-regulatory 42 RNA elements and riboswitches within 5'-untranslated regions (5'-UTR) of genes. By 43integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of 44 conditions, we establish the response of these regulators to changes in growth conditions and 45 validate several of them. Furthermore, to determine the importance of ribo-regulation by 5'-UTR 46 elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, 47 successful colonization and infection in mice suggesting that such RNA elements are potential 48 drug targets. Importantly, we show that our approach of combining high-throughput sequencing 49 with in vivo experiments can reconstruct a global understanding of regulation, but also pave the 50 way for discovery of compounds that target (ribo-)regulators to mitigate virulence and antibiotic 51 resistance. 52 53 shaping the transcriptional landscape of a wide range of pathogenic bacteria including 77 Staphylococcus aureus, Listeria monocytogenes, Helicobacter pylori, and several strains of 78Streptococci [6,[11][12][13][14][15][16][17][18][19]. Several RNA regulators have been validated and associated with 79 pathogenicity and virulence [20,21], and could be used as highly specific druggable targets 80 [22,23], however, only a select set of regulators have been targeted to date [24,25]. 81 82 Streptococcus pneumoniae is a major causative agent of otitis media, meningitis, pneumonia, and 83 bacteremia. It causes 1.2 million cases of drug-resistant infections in the US annually and results 84 in ~1 million deaths per year worldwide [26-28]. While high-resolution transcriptional mapping 85 data are available for other Streptococcus species, these studies have shown limited experimental 86 validation [17], or have focused primarily on the role of sRNAs in virulence [29]. Additionally, 87 previous studies of the S. pneumoniae transcriptome have demonstrated the presence of ncRNA 88 regulators and assessed their roles in infection and competence, however, these studies also 89 largely focused on ...

Severe Factor (F.) VIII deficiency is rare in females. We have previously reported two (unrelated) females with 1-3% F. VIII activity (Jour. Pediatr. 74:265-271, 1969). This report concerns a third Michigan female with Hemophilia A. Although this 12 year old girl’s F. VIII activity has ranged from 1-3% she has had only occasional hemarthroses. Her bleeding time is normal, her platelets aggregate normally with ristocetin and F. VIII antigen quantitation has varied from 1.02-1.40 μ/ml. In addition, platelet retention in a glass bead column has been normal. Her father has classical hemophilia A (F. VIII activity <1%, F. VIII antigen 1.61 μ/ml., normal bleeding time and normal platelet aggregation with ristocetin), while her mother appears to be normal (F. VIII activity 90-105% with corresponding values for F. VIII antigen, normal bleeding time and normal platelet aggregation with ristocetin). The child’s only sibling, a 9 year old sister has a F. VIII activity of 40-46%, F. VIII antigen of 1.40 μ/ml., normal bleeding time and normal platelet aggregation with ristocetin. While both girls are obligate carriers, one can only speculate on the reasons for the very low levels of F. VIII activity in the propositus. Although it is possible that there was a spontaneous mutation in the propositus’ maternal X chromosome, it seems likely that her 1-3% F. VIII activity reflects an extreme degree of lyonization.