title: Bacterial small RNAs in the Genus Rickettsia.
abstract: BACKGROUND
  Rickettsia species are obligate intracellular Gram-negative pathogenic bacteria and the etiologic agents of diseases such as Rocky Mountain spotted fever (RMSF), Mediterranean spotted fever, epidemic typhus, and murine typhus. Genome sequencing revealed that R. prowazekii has ~25 % non-coding DNA, the majority of which is thought to be either "junk DNA" or pseudogenes resulting from genomic reduction. These characteristics also define other Rickettsia genomes. Bacterial small RNAs, whose biogenesis is predominantly attributed to either the intergenic regions (trans-acting) or to the antisense strand of an open reading frame (cis-acting), are now appreciated to be among the most important post-transcriptional regulators of bacterial virulence and growth. We hypothesize that intergenic regions in rickettsial species encode for small, non-coding RNAs (sRNAs) involved in the regulation of its transcriptome, leading to altered virulence and adaptation depending on the host niche.
  
  
  RESULTS
  We employed a combination of bioinformatics and in vitro approaches to explore the presence of sRNAs in a number of species within Genus Rickettsia. Using the sRNA Identification Protocol using High-throughput Technology (SIPHT) web interface, we predicted over 1,700 small RNAs present in the intergenic regions of 16 different strains representing 13 rickettsial species. We further characterized novel sRNAs from typhus (R. prowazekii and R. typhi) and spotted fever (R. rickettsii and R. conorii) groups for their promoters and Rho-independent terminators using Bacterial Promoter Prediction Program (BPROM) and TransTermHP prediction algorithms, respectively. Strong σ70 promoters were predicted upstream of all novel small RNAs, indicating the potential for transcriptional activity. Next, we infected human microvascular endothelial cells (HMECs) with R. prowazekii for 3 h and 24 h and performed Next Generation Sequencing to experimentally validate the expression of 26 sRNA candidates predicted in R. prowazekii. Reverse transcriptase PCR was also used to further verify the expression of six putative novel sRNA candidates in R. prowazekii.
  
  
  CONCLUSIONS
  Our results yield clear evidence for the expression of novel R. prowazekii sRNA candidates during infection of HMECs. This is the first description of novel small RNAs for a highly pathogenic species of Rickettsia, which should lead to new insights into rickettsial virulence and adaptation mechanisms.
authors.affiliation: Department of Pathology, the University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555, USA. caschroe@utmb.edu.
authors.forename: Casey L C
authors.initials: CL
authors.lastname: Schroeder
authors.affiliation: Department of Pathology, the University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555, USA. hpnarra@utmb.edu.
authors.forename: Hema P
authors.initials: HP
authors.lastname: Narra
authors.affiliation: Department of Pharmacology, the University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555, USA. mmrojas@utmb.edu.
authors.forename: Mark
authors.initials: M
authors.lastname: Rojas
authors.affiliation: Department of Pathology, the University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555, USA. absahni@utmb.edu.
authors.forename: Abha
authors.initials: A
authors.lastname: Sahni
authors.affiliation: Department of Pathology, the University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555, USA. jgpatel@utmb.edu.
authors.forename: Jignesh
authors.initials: J
authors.lastname: Patel
authors.affiliation: Department of Pharmacology, the University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555, USA. kakhanip@utmb.edu.
authors.forename: Kamil
authors.initials: K
authors.lastname: Khanipov
authors.affiliation: Department of Biochemistry and Molecular Biology, the University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555, USA. tgwood@utmb.edu.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Wood
authors.affiliation: Department of Pharmacology, the University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555, USA. yufofano@utmb.edu.
authors.forename: Yuriy
authors.initials: Y
authors.lastname: Fofanov
authors.affiliation: Department of Pathology, the University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555, USA. sksahni@utmb.edu.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Sahni
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping; D019295:Computational Biology / Q000379:methods; D016384:Consensus Sequence; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D008969:Molecular Sequence Data; D059372:Nucleotide Motifs; D056510:Position-Specific Scoring Matrices; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012329:RNA, Bacterial; D058727:RNA, Small Untranslated; D015203:Reproducibility of Results; D012281:Rickettsia / Q000235:genetics*
chemical_list: D012329:RNA, Bacterial; D058727:RNA, Small Untranslated