3. Results
3.1 Analysis of Hfq-‐dependent sRNAs
3.1.2 General characteristics of Hpr10
Hpr10 is another Hfq-‐dependent putative sRNA that was discovered in the microarray analysis of the Δhfq mutant (Dienst et al., 2008, 2010) The coding sequence for Hpr10 is located on the chromosome upstream of slr1915 in the same orientation (Figure 18A). Hpr10 is not conserved among cyanobacteria even in closely related organisms. Its secondary structure (Figure 18B) contains a vast double-‐stranded region that suggests that it might be processed by RNase III.
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Figure 18: Hpr10 and its predicted secondary structure
(A) Schematic representation of the hpr10 locus.
(B) Predicted secondary structure of Hpr10 corresponding to the respective minimum free energy state using mfold web server.
3.1.2.1 Characterization of Hpr10 knockout, overexpression and complementation mutants
In order to investigate functions of the Hpr10 knockout (section 2.4.3), overexpression (section 2.4.4) and complementation strains were generated.
Complementation of Δhpr10 was achieved by transferring the Hpr10 overexpression plasmid in the Δhpr10 strain via conjugation. The created mutant strains were verified by Northern blot analysis using radioactively labelled Hpr10 probe (Figure 19).
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Figure 19: Northern blot verification of Hpr10 knockout, overexpression and complementation mutants
The mutants were grown on BG11 medium without copper for 7 days to induce the expression of Hpr10 in the overexpression and complementation strains. 5 µg RNA was separated on 10% PAA-‐urea gel and transferred to PVDF membrane followed by hybridization with Hpr10 probe. Hybridization with 5S rRNA was made for loading control. Transcript sizes were estimated by overlapping the pictures of the membrane with the one from the EtBr-‐stained gel containing Low Range Riboruler RNA Ladder.
The presented image was combined of the lanes cut out from the initial image of the hybridized membrane; the samples were analysed together in one experiment.
First we decided to study phototaxis behaviour of Hpr10 knockout, overexpression and complementation strains under different light conditions.
However no differences in motility of Hpr10 mutants in comparison to the WT were observed (Figure 20).
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Figure 20: Phototaxis behaviour of Hpr10 mutants (previous page)
Phototaxis assay on 0,5 % BG11 plates under normal light (NL) and red light (RL); cells were grown in the special chamber with unidirectional illumination for 7 days. Hpr10 mutants behaved like the WT under all tested light conditions. The presented image was combined of the lanes cut out from the initial image of the phototaxis plate; the samples were analysed together in one experiment.
Then we analysed the growth of aforementioned mutants and discovered a slight reduction in pigment content in Hpr10 overexpression and complementation strains when compared to the WT (Figure 21). Complementation strain of Hpr10 is more similar to the overexpression one than to the WT in its phenotype because complementation mutant was constructed via introduction of pVZ321-‐
hpr10 to Δhpr10 and expression of Hpr10 integrated in this vector is higher than the natural expression of Hpr10.
Figure 21: Phenotypical analysis of Hpr10 mutants
(A) Absorption spectra of liquid cultures of WT and Hpr10 knockout, overexpression and complementation strains grown for 8 days on BG11 without copper under normal light conditions. The spectra were normalized to chlorophyll a absorption at 685 nm and OD750nm.
(B) Phycocyanin determination in liquid cultures of WT and Hpr10 overexpression and complementation strains grown for 8 days on BG11 without copper under normal light conditions.
(C) Allophycocyanin determination in liquid cultures of WT and Hpr10 overexpression and complementation strains grown for 8 days on BG11 without copper under normal light conditions.
In order to identify targets for Hpr10 we decided to monitor change in abundance of the target mRNA by performing microarray analysis. For the
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microarray experiment whole RNA from Δhpr10 mutant cultivated till logarithmic growth phase (OD750nm 0,6) in BG11 medium (each time 2 biological replicates) was extracted. WT was taken as an equivalent. Transcripts with a log2 FC ≥1,8 (for upregulated) and FC ≤ -‐1,8 (for downregulated) were taken as significantly differentially expressed.
Table 9: Microarray results of downregulated and upregulated transcripts responsive to knockout of Hpr10. Compared to the WT.
Gene name Synonym Gene product / description FC Downregulated
slr1727-‐as1 asRNA -‐3.41
sll0022 unknown protein -‐2.53
slr1478 hypothetical protein -‐2.04
sll0019
dxr 1-‐deoxy-‐d-‐xylulose 5-‐phosphate
reductoisomerase -‐2.02
sll1639 ureD urease accessory protein D -‐1.96
sll1446 rfrL hypothetical protein -‐1.95
sll0931 hypothetical protein -‐1.93
slr1980 unknown protein -‐1.84
sll0915 pqqE periplasmic protease -‐1.82
Upregulated
NC-‐232/NC247 Hpr11 3.35
NC-‐65 located upstream from sll0306
(sigB)
2.11
sll1773 pirA hypothetical protein 2.07
slr0444-‐5’UTR aroA 3-‐phosphoshikimate 1-‐
carboxyvinyltransferase 2.03
sll1006 unknown protein 1.98
sll1851 unknown protein 1.91
sll1666-‐5’UTR dnaJ, dnaJ2, dnaJ3 DnaJ-‐like protein 1.91 slr2135 hupE, ureJ hydrogenase accessory protein
HupE 1.9
slr0449 dnr probable transcriptional
regulator 1.87
sll0609 hypothetical protein 1.86
ssl0331 hypothetical protein 1.83
sll1586-‐as1 asRNA 1.83
slr1789 unknown protein 1.81
slr1529 ntrX nitrogen assimilation regulatory
protein 1.81
sll0833 appC probable oligopeptides ABC
transporter permease protein 1.8
Microarray results showed that in Hpr10 knockout mutant 9 RNA features presented reduction in transcript quantity and 15 RNA features illustrated increase in accumulation. The most downregulated in Δhpr10 is the asRNA slr1727-‐as1. However, a significant difference in transcript accumulation of the complementary (potentially target) mRNA was not detected. The same can be said about sll1586-‐as1 that showed slight upregulation in the mutant, as its complementary mRNA was also not affected. Interestingly the most upregulated RNA feature was another Hfq-‐dependent sRNA Hpr11. It is located on the chromosome between slr1822 and slr1732 in the antisense orientation; in the hfq knockout strain Hpr11 transcript could not be detected (Schürgers, 2014). Most of the features with different transcript accumulation in Δhpr10 in comparison to the WT corresponded to unknown or hypothetical proteins and could not be linked to the hfq mutant phenotype; therefore it has been decided not to proceed with the analysis of the microarray results and focus on the direct search for RNase targets.