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All assays were conducted in biological and technical triplicate, as a minimum, to allow for statistical analysis unless otherwise stated.

3.3.1 Bacterial strains and plasmids

Our SL1344 WT, and SL1344 ∆cpxR::Kan (hereafter referred to as ∆cpxR) strains were transformed with pnlpE† (2.4.5.3). Control strains, SL1344 WT and ∆cpxR expressing empty pBAD/Myc His A, were generated in the same manner.

3.3.2 Growth curves during batch culture

For measurement of aerobic growth, LB broth or MOPS minimal media were used for batch cultures (2.3.4).

3.3.3 NlpE overexpression

SL1344pnlpE and ∆cpxRpnlpE overnight cultures were diluted 1:100 (v/v) into 50 mL LB broth supplemented with 100 µg/mL ampicillin, in biological quadruplicate. Cultures were grown at 37˚C, 200 rpm to an OD600 of 1. L-

arabinose at 0.02% (w/v) was added and cultures incubated, as previously, for four hours to allow accumulation of NlpE within the bacterial periplasm. Non-inducing controls were produced for SL1344pnlpE and ∆cpxRpnlpE. Growth conditions were identical to that described above, except for the omission of L-arabinose; dH2O was added instead. In the absence of L-

arabinose, PBAD produces very low levels of expression. Therefore, additional

negative controls were also produced, consisting of SL1344 WT and ∆cpxR harbouring empty pBAD Myc His A vectors, grown in the presence of 0.02% (w/v) L-arabinose as described for our experimental strains. Comparison of these controls to our experimental strains ensured all significant changes in gene expression during microarray analysis resulted from NlpE overexpression and subsequent induction of the Cpx system, not from the overexpression vector, the presence of L-arabinose or methodology used.

3.3.4 Microarray

Cells were collected for all samples after NlpE overexpression (3.3.3), mRNA stabilised and total RNA extracted (2.4.20). Once RNA had been quantified and quality assessed (2.4.20.2), samples were labeled with fluorescent dye Cy5-dCTP (Amersham) (2.4.22.1) and combined (1:5) with SL1344 genomic DNA (reference), labeled with Cy3-dCTP dye using Gibco Bioprime DNA labeling system (2.4.22.2). gDNA/cDNA experimental samples and standards were hybridised to an OGT array slide (8 x 15K arrays) and incubated for 60 hours (2.4.22.3). Slides were scanned on a GenePix 4000A scanner (Axon Instruments, Inc.) and quantified using GenePix Pro 7.0 software (Axon Instruments, Inc.). Normalisation of data was performed

120 using the Batch Anti Banana Algorithm in R (BABAR) (Alston et al., 2010) and further analyses using GeneSpring 7.3 Software (Agilent). A 2-fold cut off threshold was employed with a false discovery rate (FDR) of <0.05. As Cy dyes are light sensitive: reactions were kept in dark conditions during incubation and exposed to minimal light during, and post, labelling. Experiments were conducted in triplicate as a minimum.

3.4 Results

3.4.1 Overexpression of NlpE has no negative impact on the growth of S. Typhimurium SL1344 or a ∆cpxR mutant of this strain

Overexpression of NlpE is an established Cpx inducing cue (Price and Raivio, 2009, Raivio et al., 2013, Gangaiah et al., 2013, Labandeira-Rey et al., 2010). Prior to transcriptomic investigations, we wished to confirm that overexpression of NlpE from the plasmid PBAD had no detrimental impact on

growth.

SL1344pnlpE and ∆cpxRpnlpE were cultured as described in 3.3.3 to induce transcription of nlpE. The OD600 values for both strains were measured

across a ten-hour period of growth, the natural log (ln) plotted against time (Figure 9) and the specific growth rate (µ) calculated using Equation 1 (3.3.4). Growth rates for SL1344pnlpE and ∆cpxRpnlpE were µ=0.97 hr-1 and µ=1.01 hr-1 _{respectively. No significant difference in µ was seen between}

these strains in the presence of L-arabinose (0.02% w/v). Negative, empty vector controls (SL1344pBAD/Myc His A and ∆cpxR pBAD/Myc His A) cultured under the same conditions also exhibited no negative effects on growth as a result of pBAD/Myc His A introduction (data not shown).

At 0.02% (w/v), our inducer (L-arabinose) had previously been shown to induce NlpE expression in trans at sufficient levels to activate the Cpx pathway of E. coli (Snyder et al., 1995). In SL1344 this L-arabinose concentration did not have a negative impact on cell growth (Figure 9). Our SL1344pnlpE and ∆cpxRpnlpE strains were subsequently used in microarray

Figure 9: Growth of SL1344pnlpE and ∆cpxRpnlpE is unaffected by overexpression of NlpE from the PBAD plasmid. Growth curves of ln(OD600) values

collected hourly over a 10 hour period of growth, in LB broth (3.3.2). Growth rates (µ) were 0.97 hr1 _{and 1.01 hr}1 _{for SL1344pnlpE and ∆cpxRpnlpE respectively.!}

0 5 10 15 20 25 0 2 4 6 8 10 ln (O D 6 00 ) Time [h] !!! !!! SL1344pnlpE! ∆cpxRpnlpE!

studies to compare the transcriptomes of SL1344 WT and ∆cpxR under the same conditions of NlpE overexpression presented here.

3.4.2 Analysis of genes differentially expressed in SL1344 WT and ΔcpxR strains post Cpx induction

The CpxR regulated transcriptome in S. Typhimurium was evaluated using NlpE overexpression. Our genetic background for these investigations was

S. Typhimurium SL1344 (WT and SL1344ΔcpxR), chosen because no

broad-scale studies of the Cpx regulon (e.g. microarray analysis) have been conducted to date in any Salmonella serovar and SL1344 is an established

Salmonella strain where ESRs and virulence have been studied previously.

Following statistical filtering, 144 genes were differentially regulated between SL1344 WT and ΔcpxR (> 2-fold, FDR <0.05) (Appendix H; Table H1). Of these 144 genes, 77 were induced in the absence of CpxR (CpxR repressed) (Table 22), and 67 were repressed (CpxR activated) (Table 23). These 144 genes were compared to a list of all known Cpx regulon members (112 genes) generated from the current literature (Appendix G). 28 of our 144 genes had been assigned Cpx-mediated regulation prior to this study (highlighted yellow in Table H1, Table 22, Table 23) and are discussed below.

122 To better understand the regulatory networks controlled by CpxR, our 144 genes were categorised according to the Kyoto Encyclopaedia of Genes and Genomes (KEGG), using GeneSpring 7.3 Software in combination with current literature. A total of 38 categories were analysed, with changes in expression observed in all but nine of these categories (Figure 11). Raw values for the number of genes up-/down-regulated in each category are shown in Table H1. Categories of most relevance to our study will be discussed further, including genes belonging to SPI-1, where 29 genes were negatively regulated by CpxR, membrane stress and cell envelope components, which saw expression of six and 11 genes change respectively (Figure 12).

3.4.2.1 Comparison of transcriptomic data in SL1344 to current literature

Of genes identified as Cpx-regulated from current literature (Table G1), 28 (~20%) had expression levels change significantly (more than 2-fold; FDR <0.05) during our investigation. We first assessed expression of seven established E. coli Cpx regulon members (i.e. cpxA, cpxP, htrA (degP), rdoA-

dsbA, ppiA, skp (hlpA) and spy) to 1) confirm regulation of these well-

characterised genes in S. Typhimurium and 2) to confirm our methodology as an accurate means of determining Cpx-regulated gene expression (Figure 10). These genes are defined as ‘established Cpx regulon members’ because multiple research groups have confirmed their Cpx-mediated regulation (Cao et al., 2007, Danese et al., 1995, Danese and Silhavy, 1997, Pogliano et al., 1997).

As expected, cpxA and cpxP were induced significantly by CpxR in SL1344, ~22-fold and 90-fold respectively (Table 23). PpiA and HtrA, fundamental to maintaining periplasmic quality were both positively regulated by CpxR (9.86- fold and 9.22-fold) (Table 23). One of the largest transcriptional changes was observed for spy, where a 53.3-fold decrease in expression was observed in our cpxR null strain (Table 23, Figure 10). The periplasmic chaperone Spy is up-regulated by both Cpx and Bae ESRs and has had Cpx mediated regulation confirmed by multiple studies (Table H1). Another periplasmic

Figure 10: Comparison of known CpxR regulon members to those identified in