In this section the array data obtained from the wild-type and p h oP and p p k mutants is compared and contrasted. The same M T 1110 expression data was used for comparison with the respective p h oP and p p k datasets. Firstly a major similarity between the two sets was with the genes related to the PhoP regulon; in both o f the mutants pstB, pstC,
ppk, and ph o P were found to be significantly differentially expressed. Figure 4.25
includes additional genes, since even though failing significance thresholds (pfp > 0 .1 ), the expression profiles follow the patterns expected o f the other genes in the regulon.
F o ld C h a n g e p p k /W T 3 2 g 1 o o ■£. 25 0) -1 ok c «J _ o . C O 2 -3 o u_ -4 -5 T i m e (h )
Figure 4.25. Log2 average fold change o f the p si and pho genes in the ppk (A) and phoP (B) mutants with respect to the wild type, M T 1110. Genes significant at all time points with pfp < 0.12 (excluding
phoU and pstB in the phoP mutant; phoU, pstB and phoR in the p pk mutant). PhoU (SC04228) (yellow), phoR (SC04229) (orange), phoP (SCO4230) (red), pstS (SC04142) (light blue), pstC (SC04141)
(purple), pslA (SCO4140) (dark blue) and ppk (SC 04145) (green).
When comparing the same gene set with that o f the phoP mutant, an opposite pattern o f expression occurs; the p p k mutant had a greater activation o f the pho regulon genes, indicating that the p p k does have some negative regulatory influence in expression o f the PhoP regulon. In the phoP mutant both the phoR and the p h o P expressions levels were greatly under expressed, indicative o f the correct gene being mutated. The ppk gene is down-regulated in both mutants; in the pp k mutant this is to be expected as the gene is disrupted with a resistance cassette. However this decrease in p p k expression in the pho P mutant suggests that PhoP has a direct or indirect regulatory role in the activation o f p p k expression. By contrast studies by Chouayekh and Virolle, (2002) suggested that PhoP has a negative role in S. lividans, where the presence o f the PhoP maintains the inactivation o f the polyphosphate kinase in times o f phosphate limitation. If this is indeed proved correct it suggests the reverse reaction is the favoured direction in the enzymatic reaction, whereby the polyphosphate kinase acts as a dinucleotide phosphatase (Chapter 1.2.5). In E. coli the polyphosphate kinase joins inorganic phosphate into long chains o f polyphosphate; if p p k is inactivated until times o f phosphate limitation, then this cannot be the preferred pathway in the bacterial cell, as it prevents reservoirs o f phosphate to be stored during times o f phosphate abundance. A
3
As the phosphate becom es limited, actinorhodin production is induced, along with the
phoR P two com ponent system and pstSCAB. Figure 4.25b illustrates the effect o f
deleting phoP , the response regulator in the two com ponent system; both phoP and
phoR are dow n-regulated alongside the p p k and the entire pstSC A B operon. Therefore it
can be concluded from the removal o f phoP , that phoR, p p k and the pstSC AB operon are positively regulated by the PhoP.
The ram S gene is dow n-regulated in both the p h oP and the p p k mutant, the expression profile in both is very sim ilar w ith a pfp <0.001, indicating this result is highly significant. I f this gene is affected in both mutants, does this m ean it is in response to phosphate lim itation or perhaps p p k induction or polyphosphate levels? It was previously shown that an interruption in the phoP gene decreases the expression o f p p k to a level seen in the p p k mutant, suggesting the effect on ram S m ight be attributable to the level o f p p k expression. N utritional limitation could be a factor, but as was shown in Chapter 3, glucose and am monium levels were not limiting factors only levels o f phosphate. Perhaps it could be due to a phosphate limitation, how ever as illustrated in Figures 3.9a and 3.10, the p p k m utant is slightly delayed in phosphate limitation, so the expression profiles o f ram S would be expected to reflect this. The ram operon in S.
coelicolor is associated w ith differentiation and the production o f aerial hyphae (Keijser
et al., 2002; K odani et al., 2004); this process has been studied in solid agar and static liquid cultures but not in shaking liquid cultures (van K eulen et al., 2003). Could this be an explanation o f the down regulation o f the ram S gene, where the function is unnecessary in liquid cultures such as those in the fermenter? However, the ramS is down-regulated w ith respect to the wild-type, in both the phoP and p p k mutants. The wild-type was also grown in liquid culture, in exactly the same m edia and conditions as the mutants. Therefore, liquid cultivation can not be the contributing factor. Additionally w hen com paring this data w ith the S. lividans agar plate study (see Chapter 5), ram S expression was constant between 31 h and 38 h, after which a rapid decline in expression occured, indicating that once the aerial hyphae had crossed the air w ater interface, this protein was no longer required and expression levels were repressed.
W hen com paring the expression profile o f the act genes in the p p k m utant (Figure 4.25) to that o f the p h o P m utant (Figure 4.10), a marked difference can be seen. The phoP m utant overproduces actinorhodin, w ith a peak at 42 h, a dip at 46 h but then a plateau o f consistently high expression from 60 h onwards. W ith the p p k mutant, a steady reduction o f expression occurs from 31 h until 46 h after w hich there is a slight recovery and increase in expression. Even so the expression level is far more reduced than the p h oP mutant. These results are consistent w ith the antibiotic assay results w hich dem onstrated that in com parison w ith the wild-type, under the same growth conditions and media, the p p k m utant under produces actinorhodin and the p hoP mutant overproduces.
Independent validation o f selected genes identified from the transcriptomic analysis provides confidence in the differentially expressed gene lists produced in this study. PhoP has, not surprisingly been shown to have a regulatory role in controlling the pho regulon genes. O f interest is the finding that m utation o f p h oP influences a large num ber o f other genes, including those involved in nitrogen metabolism. In addition a new tw o com ponent system and associated protease has been highlighted from the
p h oP m utant analysis, possibly repressed by the protein or activated as a secondary
system for coping w ith phosphate starvation when the original system is non functional. The polyphosphate kinase seems to have a less straight forward pathway o f regulation. The inactivation o f the enzyme, causes changes in a variety o f pathways and interestingly the repression o f actinorhodin production is confirm ed by the microarray data.
Further w ork needs to be conducted on these mutants, selecting some o f the more interesting differentially expressed genes for follow up m utational studies. The transcriptional analysis itself could be made more statistically significant with at least one additional biological replicate and a detailed comparison to array data from cultures grown under phosphate replete conditions.