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

Abstract

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.

Figure 1.

Genome-wide identification and characterization of B. burgdorferi transcription start sites (TSSs). RNA was isolated from log phase B. burgdorferi clone B31 A3, and treated with and without TAP (tobacco acid phosphatase). (A) Schematic classification of the 6042 TSSs relative to the genome organization. Circled numbers indicate TSSs for each category. The maximum nucleotide distances from the 5′ and/or 3′ of annotated sequences, shown as black arrows, for a particular category of TSS are indicated in red. (B) Graphical representation of the TSS classifications. as, antisense; o, orphan; p, primary; s, secondary; i, internal. (C) Nucleotide frequency at the −100, −1, +1 (TSS) and −1 nucleotide positions. A, adenine, T, thymine, G, guanine, C, cytosine. (D) Consensus motif for promoter regions upstream of primary TSSs. The nucleotide sequences from −40 to +1 of the 321 identified primary TSSs were analyzed by MEME 4.11.2. (E) Distribution of 5′ untranslated regions (UTR) lengths among uniquely classified primary and secondary TSSs. The data are shown as the percent of mRNA sequences with a 5′ UTR of each bin length. 0–10 nucleotides (red bar), bin size of 20 nucleotides, where the number shown is the middle length in each bin (black bars). (F) Consensus ribosome binding motif for uniquely classified, primary 5′ UTRs. MEME 4.11.2 was used to analyze UTR sequences ranging from 10–293 nts in length for a conserved motif, which was found on average to begin 8 nts upstream of the annotated start codon. (G) Northern blot analyses validate a long 5′ UTR. Total RNA was extracted from mid-log phase spirochetes and separated by denaturing formaldehyde–agarose gel, blotted to nylon membranes and probed with ³²P-labeled complementary probes, indicated by red and black boxes. Genomic context of ORFs BB_0240-BB_0243 (wide black arrows), the primary TSS (green bent arrow), putative transcripts and their position on the Northern blot (broken line, green arrows and marked with designated symbol) are indicated. Marker sizes in nucleotides are indicated to the left of each blot.

Figure 2.

B. burgdorferi in vivo expression technology (BbIVET)-identified sequences with associated TSSs. (A) Schematic representation of BbIVET associations with 5′RNA-seq TSSs. Brackets designate the parameters for the association. Relative orientation of the genome region (wide black bar with white arrows), Bbive sequence (thin black arrow) and TSS (orange bent arrow) are indicated. The minimum and maximum Bbive-TSS association distances were defined as 20 nts from the 5′ end and 6 nts from the 3′ end of a Bbive sequence. (B) Categorization of Bbives that associate with 5′RNA-seq TSSs.

Figure 3.

Validation of BbIVET-associated RNA transcripts. (A) Deep-sequencing screen shot for a Bbive-internal TSS, displaying only the sequenced 5′ nucleotide, of overlaid biological replicates treated with (TAP+) and without (TAP−) tobacco acid pyrophosphatase. Read count ranges are shown in the upper left of each frame. The chromosome nucleotide coordinates, relative orientation of the BB_0370 ORF (wide black bar), Bbive45 sequence (thin black arrow), internal TSS (blue bent arrow), processed 5′ ends (scissors), putative transcripts (broken line arrows), Northern probe locations (black and red boxes) and luciferase fusion regions (brackets) are indicated. The predicted transcripts of interest are marked with an asterisk. (B) Northern blot analyses of the Bbive45 internal TSS, as described in the Figure 1 legend. Probes located upstream (red) and downstream (black) of the putative internal TSS, are indicated. Marker nucleotide sizes are indicated to the left of the blots. (C) Bbive45 promoter activity and specificity. B. burgdorferi clones harboring specific promoter fusions were grown to mid-log phase, and incubated with 750 μM D-luciferin. Relative luciferase units (RLU) were normalized to cell density by OD₆₀₀. Data represent the mean ±SD from three biological replicates shown in log scale and were analyzed relative to pJSB161 with the two-tailed Student's t-test. Unless otherwise indicated all fusion constructs demonstrated significantly greater RLUs than the promoterless control, pJSB161 (P ≤ 0.01). n.s., not significantly greater RLU relative to pJSB161. (D) Schematic representation of Bbive luciferase fusions. The bracket designates the sequence selected for promoter fusion to luciferase in pJSB161. Relative orientation of the genome region (wide black bar with white arrows), 5′ boundary of Bbive sequence (orange line) and TSS (orange bent arrow) are indicated. (E) A variety of Bbives with associated TSSs have promoter activity in culture. Spirochetes containing control promoters (flaBp, ospCp and ospAp) and specific Bbives, fused to luciferase, were grown to mid-log phase, and analyzed as described above.

Figure 4.

Culture-expressed Bbive-TSSs demonstrate activity in the mammalian host. C3H/HeN mice infected intraperitoneally with 1 × 10⁵B. burgdorferi containing promoterless pJSB161, control promoters (flaBp, ospCp or ospAp) or specific Bbive luciferase fusions as indicated (left). Mice were injected intraperitoneally with 150 mg/kg D-luciferin 7 and 10 days post infection and imaged with the IVIS^TM 50 Imaging System (IVIS) using a 5 min exposure. Images were normalized to the same p/s range of 1.73e4 to 1.128e5 and displayed on the same color spectrum scale (right). Data are representative of two independent biological replicates. asTSS, antisense TSS; p/iTSS, primary or internal TSS; iTSS, internal TSS.

Figure 5.

Novel B. burgdorferi RNA transcripts expressed during murine infection. (A) A subset of Bbives that lack or have low read count associated TSSs demonstrate low promoter activity in culture. Bbive sequences were fused to luciferase in pJSB161 and measured for luciferase activity, as described in Figure 3 legend. Data represent the mean ± SD from three biological replicates shown in log scale and were analyzed relative to pJSB161 using the two-tailed Student's t-test. Unless otherwise indicated all fusion constructs demonstrated significantly greater RLUs than the promoterless control, pJSB161, (P ≤ 0.01). n.s., not significantly greater RLU relative to pJSB161. (B) Bioluminescence of Bbive sequences in the murine host. C3H/HeN mice infected intraperitoneally with 1 × 10⁵B. burgdorferi containing controls and specific Bbive luciferase fusions, analyzed by IVIS as described in Figure 4 legend. Symbols indicate specific controls for each time point. Data are representative of two biological replicates. Note that the same color spectrum scale is used between figures.