Characterization of <i>cycA</i> merodiploid BCG and <i>M. smegmatis</i> strains.

<p>(<b>A</b>) Sequence of the SNP containing region in <i>cycA</i> from BCG-Pasteur::I425 and BCG-Pasteur::pYUB412 clones from the 5′ end. (<b>B</b>) CFUs of BCG-Pasteur::I425 (white bars) and BCG-Pasteur::pYUB412 (black bars) 4 days after treatment with indicated concentrations of DCS. (<b>C</b>) CFUs of BCG::I425 (solid squares) and BCG::pYUB412 (solid diamonds) obtained over time upon exposure to 32 µg/mL DCS. (<b>D</b>) CFUs of <i>M. smegmatis</i>::I425 (white bars) and <i>M. smegmatis</i>::pYUB412 (black bars) 24 hours after treatment with indicated concentrations of DCS. (<b>E</b>) CFUs of <i>M. smegmatis</i>::I425 (solid squares) and <i>M. smegmatis</i>::pYUB412 (solid diamonds) obtained over time upon exposure to 200 µg/mL DCS. Data points and error bars are means ± standard deviations. Statistically significant differences are denoted by asterisks (*, P<0.05; **, P<0.01). Representative of 3 independent experiments performed in duplicate.</p

Susceptibilities of <i>M. smegmatis</i> strains overexpressing <i>cycA</i> alleles to DCS.

<p>(<b>A</b>) CFUs of recombinant <i>M. smegmatis</i> strains after 24 hours of treatment with indicated concentrations of DCS. (<b>C</b>) CFUs of recombinant <i>M. smegmatis</i> strains obtained over time upon exposure 200 µg/mL DCS. <i>M. smegmatis</i> harbouring pMD31 (black bars), pMDcycA_Mtb (white bars), pMDcycA_Mbov (light-gray bars) and pMDcycA_BCG (dark-gray bars). Data points and error bars are means ± standard deviations. Statistically significant differences are denoted by asterisks (*, P<0.05). Representative of three independent experiments performed in duplicate.</p

Analyses of CycA amino acid sequences.

<p>(<b>A</b>) Partial CycA amino acid sequence alignment of various mycobacteria (the conserved G122 residue is indicated by the arrow). (<b>B</b>) 2-dimensional topological representation of <i>M. bovis</i> CycA with the G122S mutation found in all BCG (circled) in the extracellular loop (boxed) between the 3^rd and 4^th trans-membrane helices from the amino (N) terminus.</p

DNA and bacterial strains used in this work.

<p>DNA and bacterial strains used in this work.</p

IsomiR abundances and variant representation in different datasets.

<p>A) IsomiR normalised counts compared between 1) fresh (-80°C/<1 year storage) and biobanked (-20°C/>10 year storage) bovine serum, 2) serum and macrophage (both bovine) and 3) bovine and human serum. B) Classification scheme for isomiRs using sRNAbench is hierarchical. <i>exactNucVar</i> means single nucleotide changes to the canonical sequence, most probably due to sequencing errors. <i>mv</i> indicates shifted sequences. non-templated addition is enzymatically addition of a nucleotide to the 3’ end and is given priority by sRNAbench since these changes may be of biological relevance. C) Plot shows the counts of dominant isomiRs categorised by class. The general trend of dominance is the same across all datasets, including non-serum.</p

Comparisons between miRNAs from fresh and biobanked serum.

<p>A) Fractions of small RNA identified in both fresh (-80°C/<1 year storage) and biobanked (-20^°C/>10 year storage) according to category using Bowtie mapping. UMD3.1 denotes mappings to the reference genome. miRNA fractions are shown inset in detail. Note these values slightly underestimate the total miRNAs present since they only include miRBase hairpins and not novel content. B) Overlap between fresh (-80°C/<1 year storage) and biobanked (-20°C/>10 year storage) serum core miRNAs and a bovine macrophage dataset. C) Comparison of top 8 miRNAs in fresh (-80°C/<1 year storage) and biobanked (-20°C/>10 year storage) samples shows large differences in the top 2 most abundant miRNAs. Counts are the means of normalised value per sample and error bars show the standard deviation over all n samples. For fresh samples n = 24, for biobanked samples n = 57. D) Correlation between log mean normalised counts for the same miRNA in fresh (-80°C/<1 year storage) and biobanked (-20°C/>10 year storage) datasets. Error bars indicate the variance over all samples of mean read count.</p

isomiR abundance patterns compared between fresh and biobanked serum.

<p>A) Relative abundances of isomiRs with a single dominant form in the fresh (-80°C/<1 year storage) dataset but not in the biobanked (-20°C/>10 year storage) data. Note that many of the low abundance forms are not present in the biobanked data (green bars). B) Relative abundances (shown as fraction of total) of some miRNAs with multiple sub-dominant isomiRs compared between fresh and biobanked year samples.</p

Canonical and dominant isomiR differences.

<p>A) bta-miR-22-3p total abundance profile over time (left) and for 2 isomiRs (right), both are shorter 3’ variants. Analysing these isomiRs individually allows them to be detected as being differentially expressed over time. B) bta-miR-22-3p isomiR relative abundances. The canonical form, labelled <i>exact</i>, is only fourth most abundant. C) The canonical and dominant isomiRs differ in >50% of cases. Shown in the plot are the corresponding percentages between the 2 isoforms, when different. (By definition <i>dominant</i> means highest percentage of reads). For points in the top left of the plots, the actual canonical form is insignificant. Note the lower number of points for the 10–15 year (CVI) dataset.</p

Monitoring of the MAP experimental infection.

<p><b>(A)</b> Detection of anti-MAP antibodies in serum via ELISA across 49 months of the time-course. Six individual experimentally infected cattle (in red A-F) and six individual naturally infected cattle (in green G-L) are represented. Faecal culture data for <b>(B)</b> experimentally infected and <b>(C)</b> naturally infected animals at monthly intervals. 0 = negative, 1 = 1 cfu/ agar slant, 2 = <50 cfu/agar slant, 3 = <100 cfu/agar slant and 4 = >100 cfu agar slant. C = fungal contamination, resulting in an inability to accurately determine MAP cfus.</p

Normalised read variation for differentially abundant miRNA over 5 time points.

<p>Normalised reads are shown for distinct miRNA at the 0, 6, 43, 46 and 46-month time points. Note the x axis time interval is not to scale.</p