Influence of Hyperosmotic Shock on the Expression Levels of Repeat Associated Genes

Among the repeat associated genes in Xcc we noticed various genes in connection to osmoadaption, the most obvious ones being kdpC, kdpD and osmC (see Introduction Chapter 1.6).

Weber and Jung carried out a profiling of early osmostress dependent gene expression in E. coli using a DNA macroarray after treating the bacteria with 0.4 M NaCl for 9 min (221). In total 152 genes were detected that showed changed expression after shock compared to before. Genes were considered differentially regulated when the change in expression level was greater than 1.4 fold.

We found a number of the genes that were differentially regulated in E. coli to be homologs of repeat associated genes in Xcc. Among the upregulated genes are leuC (#150), osmC (#120) and lpxA (#073, #074). Repeat associated genes among the downregulated genes are leuD (#150), proA (#101), ftsZ (#041, #042), pyrB (#120), atpC (#020, #021), gltB and gltD (#003, #004), cysD (#137, #138), cysI and cysJ (#140, #141), rplI and rpsR (#083), ileS (#058) and trmD (#060). The repeat patterns associated with kdpC/kdpD and osmC/pyrB belong to the group of the prominent members of GGGAATC repeats.

High osmolarity also increases negative supercoiling in the bacterial genome (see Introduction Chapter 1.6) (215,224,226). Cheung et al. reported that an increase in negative supercoiling is crucial for the induction of many genes upregulated during hyperosmotic conditions in E. coli (225). Generally, negative supercoiling in increased upstream of the RNA polymerase whereas positive supercoiling is found downstream of the enzyme during transcription (227-229).

Interestingly, Zhang et al. reported G-quadruplex formation in response to transcriptional activity at remote downstream locations as the mechanical torsion imposed by supercoiling upstream of the polymerase is propagated through the double helix. They proposed that G-quadruplex formation at distal sites may work as a relay of the transcriptional activity (230). As previously mentioned i-motif formation may also be induced by negative supercoiling (62,104).

Taken together the finding of GGGAATC patterns in proximity to genes involved in osmoadaption, increasing K⁺ levels and negative supercoiling conditions during hyperosmotic challenges and G-quadruplex formation of 5’-(GGGAATC)3GGG-3’ in KCl solution prompted us to investigate the expression levels of repeat associated genes under osmotic shock conditions to check for G-quadruplex dependent regulation of transcription.

So far most studies on osmostress have been carried out in E. coli and little data was available of the response of Xcc to hyperosmotic conditions. Qian et al. treated Xcc with 1 M sodium chloride for 10 min during a salt stress experiment and 40% D-sorbitol for 40 min as osmotic challenge to impose lethal environmental conditions. Therefore conditions applied in experiments with E. coli or Salmonella were taken as reference points (209,221,223). 0.3 M sodium chloride was initially chosen as stress inducer, however osmoshock controls genes had shown a reduced response to

D-sorbitol were used in later experiments. Experimental procedures are detailed in Chapter 7.20-7.27. Briefly, Xcc was grown in minimal medium M9 at 28°C to an OD600 = 0.3. Uninduced cells were taken as controls. Osmotic shock was induced by addition of prewarmed sucrose (final concentration 0.6 M) or sorbitol (final concentration 20% (w/v)) in M9. Cells were osmotically challenged for 7 min, 15 min and 30 min and then collected by centrifugation. RNA was isolated using the RNeasy mini Kit. After treatment with DNase I for removal of genomic DNA reverse transcription was carried out using SuperScript III Reverse Transcriptase. cDNA was analyzed by semi-quantitative Real-Time PCR (qPCR) with Phusion HotStart II Polymerase. RNA levels were determined relative to 16S rRNA as described in Chapter 7.27, values of four biological replicates were averaged. ct values (cycle threshold, i.e. the number of cycles determined for fluorescence to exceed background level) of 16S rRNA were comparable for all samples and are shown in Figure 58. Induction ratios were obtained by dividing the normalized levels of mRNAs at each time point after the osmotic shock by the average of the normalized levels of those mRNAs before the osmotic shock (control). A threshold of a fold change of 3 was defined for differentially expressed genes.

Figure 40: Relative RNA Levels of Hyperosmotic Shock Controls

Expression levels during hyperosmotic shock are shown for control genes relative to 16S rRNA over a time course of 30 min (0 min = control, dark blue; 7 min blue, 15 min cyan, 30 min green). Uninduced cultures were used as controls.

A: Expression levels of cells treated with 20% sorbitol. B: Induction ratio after sorbitol treatment. C: Expression levels of cells treated with 0.6 M sucrose. D: Induction ratio after sucrose treatment. 0.6 M sorbitol. ostB, proP and osmC show upregulation under both conditions. Errors represent standard deviation of four biological replicates.

The following genes were chosen as controls: kup encodes a low affinity K⁺ transporter. It is constitutively expressed in E. coli and its level should not vary greatly under hyperosmotic shock (208,209). kdpA encodes a component of the high affinity KdpABC K⁺ transporter that is induced under high osmolality of the medium in E. coli (210,211). Furthermore one of the prominent repeats is located between kdpC and kdpD. In E. coli kdpC and kdpA are transcribed together as they are located within the same operon kdpFABC, levels of kdpA and kdpC are therefore expected to show comparable levels in Xcc. Trehalose-6-phosphate phosphatase is encoded by ostB in Xcc (Note: annotated as otsB in E. coli, Salmonella, Xcc 8004, but as ostB in KEGG and NCBI for Xcc ATCC 33913), proP encodes a transport system for proline, osmC the osmotically inducible protein OsmC, all have been shown to be upregulated in E. coli or Salmonella (206,217,221,222,290).

Shock with sorbitol and sucrose had similar effects on the control genes. ostB and osmC show more than 4-fold upregulation under both conditions (Figure 40). For ostB a sudden increase occurs between 7 and 15 min in sorbitol treated cells, and slightly delayed in sucrose treated cells. In contrast upregulation of proP and osmC occurs gradually. Levels of proP reach a maximum around 15 min and then decline again. RNA levels of kup increased only minimally. kdpA did not show upregulation as expected, however RNA levels showed very high ct values and may be difficult to evaluate in comparison to the very abundant 16S rRNA. Profiling of early osmostress dependent gene expression in E. coli after treating the bacteria with 0.4 M NaCl for 9 min using a DNA macroarray also had not shown kdpFABC as upregulated although the threshold for differentially regulated genes had only been set at 1.4-fold instead of 2-fold as suggested by the manufacturer of the chip. Retesting by Northern Blotting then showed 6-fold upregulation of kdpFABC (221).

Detection in qPCR relies greatly upon amplification efficiency of the primer pair used; while the primers used for kdpA only showed one product in agarose gel electrophoresis and melting analysis (data not shown), amplification efficiency may be too low to adequately assess RNA levels using qPCR. Balaji et al. examined the timing of induction of osmotically controlled genes by exposure to in Salmonella by qPCR. The time course for induction of proP, ostB and osmC is in accordance with the one they describe for osmotic shock with 0.3 M sodium chloride and 0.6 M sucrose (see Introduction Chapter 1.6). In addition they also note that levels of kdp were reduced in sucrose challenge in comparison to shock with sodium chloride (222). In summary, hyperosmotic shock of Xcc was achieved under both applied conditions and gene expression responses and time course of the control genes are in accordance with those reported in the literature for other bacteria.

Repeat associated genes were chosen for an initial test included genes that had been shown to be differentially regulated upon osmotic shock in E. coli: osmC, pyrB, atpC, gltB and gltD. Other candidates include the genes encoding flagellar proteins flgE and flgF, genes encoding components of the type II secretion system xpsE and xpsF and genes encoding Holliday junction resolvases ruvA and ruvB. In addition, genes associated to the longest repeats xcc0513, prmA, osmC, pyrB, cebR,

suc1, xcc0176 and cls. pdeA was chosen because a repeat sequence is overlapping with its stop codon. Furthermore repeat associated genes were chosen to include inverted repeats and single long repeats and different orientations of genes relative to the repeats. All repeats chosen were putative G-quadruplex forming sequences. Both neighboring genes were analyzed except in the case of ruvC for which primer design proved to be difficult, instead ruvB was chosen. flgF was assayed with two different primers as an additional control.

Relative expression levels of treatment with sucrose are shown in Figure 41 and Figure 42 for sorbitol. Similar trends could be observed for sucrose and sorbitol treatment, the majority of genes were downregulated rather than upregulated, generally strongest effects were observed after 30 min.

Figure 41: Relative RNA Levels of Repeat Associated Genes after Hyperosmotic Shock with 0.6 M Sucrose A: Expression levels during hyperosmotic shock are shown for repeat associated genes relative to 16s rRNA over a time course of 30 min (0 min, control, dark blue; 7 min blue, 15 min cyan, 30 min green). Uninduced cultures were used as controls. Cells were treated with 0.6 M sucrose. Errors represent standard deviation of four biological replicates.

Respective induction ratio is shown in B as multiples of control, dashed lines mark threshold of 3-fold up- or downregulation.

Figure 42: Relative RNA Levels of Repeat Associated Genes after Hyperosmotic Shock with 20% sorbitol A: Expression levels during hyperosmotic shock are shown for repeat associated genes relative to 16S rRNA over a time course of 30 min (0 min, control, dark blue; 7 min blue, 15 min cyan, 30 min green). Uninduced cultures were used as controls. Cells were treated with 20% sorbitol. Errors represent standard deviation of four biological replicates.

Respective induction ratio is shown in B as multiples of control, dashed lines mark threshold of 3-fold up- or downregulation.

During sorbitol treatment osmC, that was also used as osmoshock control, showed the strongest upregulation of up to 4.5-fold. For sucrose treatment the strongest upregulation was observed for suc1, a sugar transporter, with up to 12-fold. In contrast suc1 was not differentially expressed under sorbitol treatment. flgE, encoding the flagellar hook protein FlgE, and flgF, encoding the flagellar basal body rod protein FlgF, all showed greater than 3-fold downregulation. The effect was stronger with sorbitol shock. Here, 6-fold, 8-fold and 13-fold downregulation were observed for flgE, flgF, flgF-2, respectively. atpC, atpD, gltD, kdpD, xpsE, and pdeA all showed minor downregulation of 2 - 3-fold. Downregulation had also been observed for atpC and gltD in E. coli (221). The remaining genes were not found to be differentially expressed under the set threshold.

As observed for kdpA expression levels for kdpC were again very low under both conditions.

Changes in expression level for flgF were assayed with two primers. While both primer pairs showed the same trend of downregulation, relative mRNA levels and therefore calculated induction ratios differed, this is due to different primer efficiencies. Changes in expression levels were calculating approximating an amplification efficiency of two, for accurate determination of mRNA levels in the future amplification efficiency will have to be determined for each primer pair.

However the approximation made is sufficient to gain an overview of the responses to osmotic shock in this preliminary experiment. In summary, the majority of repeat associated genes were not differentially regulated under the set threshold; the general trend was a downregulation of gene expression.

When taking into account only the genes associated with single and primarily long repeats (Table 12) there are four genes with repeats located in close proximity upstream of the ORF on the anti-sense strand: xcc0513, osmC, xcc0178 and xpsF (◄R). Strong upregulation was observed for osmC, but no differential regulation for xcc0513, xpsF and xcc0178.

Table 12: Induction Ratios of Genes Associated with Long Repeats

Table shows the induction ratios of genes associated with long repeats after 30 min osmotic shock. Location and distance of the ORF relative to the repeat is shown (green arrow). More than 3-fold upregulated genes show induction ratio greater 3 (red bold), more than 3-fold downregulated genes show induction ratio less than 0.33 (blue bold). us = upstream, ds = downstream

In this set there are also three genes with repeat sequences located upstream of the ORF on the sense strand: prmA, yeiM and ruvB (R►). None of these were differentially regulated. There does not seem to be a general trend with regard to repeat location and orientation.

Interpretation of the results obtained for genes associated with inverted repeats (Table 13) is more difficult as the repeats are always found on the coding and non-coding strand within the same intergenic region. Generally, only downregulative effects were observed for this set irrespective of the orientation and location of a gene to a repeat. flgE and flgF showed the strongest downregulation. The inverted repeat is located downstream of flgF and upstream of flgE, operon prediction had yielded contradictory results as to whether these genes are transcribed together (Table 52).

Table 13: Induction Ratios of Genes Associated with Inverted Repeats

Table shows the induction ratios of genes associated with inverted repeats after 30 min osmotic shock. Location and distance of the ORF relative to the repeat is shown, respective gene is marked by green arrow. More than 3fold upregulated genes show induction ratio greater 3 (red), more than 3-fold downregulated genes show induction ratio less than 0.33 (blue). us = upstream, ds = downstream

gene induction ratio

The strong downregulation observed for both suggests they are part of a polycistronic mRNA.

Furthermore, in the sequencing data obtained from Xac the homologous genes were found to be located on one transcript.

Holder and Hartig recently reported the effects of G-quadruplexes inserted upstream of ORFs within the promoter region or 5’UTR. The observed regulatory effects were strongly dependent on strand orientation and the exact location of the G-quadruplex. While inhibitory effects were observed anti-sense to the core promoter region, the same sequences inserted after the -10 region had activating effects. Up- and downregulation of gene expression was observed upon insertion of G-quadruplexes on the sense strand in or near the ribosomal binding site (78). To gain a better insight into the possible modes of action a potential G-quadruplex may have the respective repeat associated genes will have to be further analyzed with repect to putative promoters and ribosomal binding sites and the data compared to the changes in gene expression observed. Overall broad evidence for a gene regulative function of GGGAATC repeats during osmotic shock could not be observed. The number of genes tested in this initial screen is small, but in particular concerning longer repeats 6 out of the 12 longest repeats (see Table 4) were analyzed. Still a general function as gene regulators cannot be ruled out from this preliminary experiment as hyperosmotic shock is only one of many different triggers possible. For instance due to the differences in GGGAATC patterns between Xac and Xcc, Xcc may also be subjected to growth in hypersensitive response eliciting medium. Control primers for such an experiment have already been designed (Table 19).

In addition other environmental stressors may be tested such as starvation, heat or cold shock, oxidative stress or stringent reponse (291-294).

Nevertheless osmC may be an interesting target for further studies. Now that sucrose and sorbitol treatment have been shown to be adequate inducers of osmotic shock in Xcc, chemical probing (DMS footprinting) could be applied during an osmotic upshift to assess whether G-quadruplex formation takes place. Furthermore observing gene expression changes under hyperosmotic shock in dependence on the number of repeat units might help gain an insight into the physiological role of the respective repeat pattern. Obtaining genomic mutations in Xcc is not easily done, instead reporter gene constructs consisting of the whole repeat containing upstream region of osmC in front of an eGFP or β-galactosidase reporter gene on a plasmid could be constructed. Repeat length could then be varied with standard cloning methods and responses to osmotic shock assessed in E. coli and Xcc. Finally, one could test whether incubation with G-quadruplex binding small molecules such as N-methyl mesoporphyrin (NMM) enhances the gene regulative effect observed under hyperosmotic conditions (137). MIX, an unmethylated analogue of NMM, which is reported to not bind to G-quadruplex motifs could be used as a negative control.

Small molecule treatment is applicable to both the natural situation and a reporter system on plasmid.

The strong induction of suc1 during osmotic shock with sucrose seems to be unrelated to the repeat found in close proximity. Firstly, the same upregulation is not observed when the cells were treated with sorbitol, secondly the repeat is located downstream of the ORF, and Holder and Hartig found no effect of G-quadruplex forming sequences on gene expression when inserted in the 3’UTR (78). suc1 had also been upregulated when Xac was grown in XVM2 medium, which contains 10 mM sucrose and fructose (285). Upregulation is most likely due to the presence of the sugar in the medium, this emphasizes that our method of detection generally is applicable and may be used to study the effect of other triggers.