Polyphosphate kinase studies

The pho regulon and phosphate limitation is a well researched topic, in a multitude o f bacteria including S. coelicolor. This has made the phoP experiments much easier to study and link w ith published data. However, the polyphosphate kinase study has not been so straightforward. The num ber o f genes passing the significance threshold was far greater in the S. coelicolor study than in the p h o P data o f the same species, however as noted previously the S. lividans data was far more com pressed and genes discovered between the two species were not in as much agreem ent as w ith the phoP mutant. There was one striking result, p p k was down-regulated in all studies w ith both Streptomyces species. This was as expected in two o f the studies since they were performed using the

p p k mutant, hence this data confirm ed the inactivation o f the p p k gene. However p p k

was dow n-regulated in both o f the phoP mutants, one grown on the minimal liquid media and the other on the com plex m edia agar plate study, com pletely different growth mechanisms but still generating a reduction o f the p p k expression. These results suggest that p p k m ust belong either directly or indirectly to the p h o regulon, since inactivation o f phoP caused repression o f the ppk. This finding was consistent with those found previously by Ghorbel et al. (2006b) where western blot analysis clearly demonstrated that the p p k induction was greatly reduced in the phoP m utant o f S. lividans. However, these results should be further verified, since the position o f the insertion cassette could have affected transcription and therefore subsequent results.

The polyphosphate kinase seemed to affect how the phosphate was taken up and m etabolised in the cell o f S. coelicolor. In the p p k m utant cultures on both phosphate lim ited and replete cultures, phosphate rem ained in the m edia for longer and in the case o f the phosphate replete cultures much higher levels o f phosphate were present than in both the p h o P m utant and the wild type strain media. Polyphosphate kinase has numerous functions, w ith the reversible polym erisation o f phosphate from ATP into long chains o f polyphosphate (Ghorbel et al., 2006), used as storage for times o f phosphate limitation. A possible reason for the reduced uptake o f phosphate could be the lack o f the kinase enzym e meaning a conversion o f phosphate and storage o f polyphosphates will not occur. H owever the polyphosphate kinase cannot be the only such enzyme o f its kind and to clarify the reaction, the polyphosphate reservoirs from w ithin the cell should be studied, w hich alongside the phosphate assay would give a greater understanding into the whole phosphate uptake process. Consistent with previous results found by Ghorbel et al. (2006a) the p p k m utant had elevated expression o f the phoR and pho P (60 h and 81 h) in S. coelicolor in com parison to the wild type. The S. lividans concurs w ith both phoR and phoP , alongside p h o U being up-regulated in the p p k mutant, hence agreeing w ith the suggestion there is an additional phosphate signal am plified in the p p k m utant (Ghorbel et al., 2006a).

Elevated actinorhodin production in the S. lividans p p k m utant was also consistent with previous results (Chouayekh and Virolle, 2002; Ghorbel et al., 2006b), the inactivation o f the p p k induced early onset o f undecylprodigiosin and actinorhodin the latter not usually produced in the parental strain. H owever the same result does not apply to the

p p k m utant in S. coelicolor, inactivation caused very low levels o f antibiotics. In the

study by Ghorbel et al. (2000b) the p p k m utant o f S. lividans was found to have elevated levels o f glucose uptake, w hen compared to the parental strain, suggested to be the reason for the antibiotic synthesis. The proposed theory suggested the p p k m utant is starved o f its ATP regenerating enzyme (polyphosphate kinase), altering the internal energy charge o f the cell and to compensate an activation o f the central metabolic pathways occurs, inducing uptake o f glucose and phosphate and therefore reducing cofactors w hich could additionally be used in antibiotic synthesis.

W hile this theory works with the study they performed in S. lividans, the opposite results found in the p p k m utant o f S. coelicolor confuse the matter. Metabolites were taken and analysed through the entire growth curves for each o f the strains and replicates perform ed (Chapter 3.6). The p p k m utant was found to have lower levels o f both undecylprodigiosin and actinorhodin, in com parison to the w ild type and the phoP mutant. W hen the external metabolites o f glucose and am m onia are studied in more detail, glucose consum ption is fairly similar between the p h oP and the wild type, but greatly reduced in the p p k w ith higher concentrations o f glucose rem aining in the media at the latter tim e points, opposing those results found in the S. lividans study, perhaps explaining the difference between the antibiotic synthesis o f both species. Phosphate levels are in lim itation in all three strains, how ever the p p k m utant consumes phosphate at a far slower rate than the other two strains. In the non-producing strain, S. lividans,

p p k inactivation induces antibiotic synthesis, phosphate and glucose uptake; in the

producing strain, S. coelicolor, p p k inactivation represses antibiotic production, reducing the uptake o f both glucose and phosphate. To clarify the difference was not due to solid versus liquid media, as all cultures o f the parental strains and p p k mutants o f both S. coelicolor and S. lividans were perform ed on com plex liquid media (Figure 3.12), w hereby the same result occurred. Transcriptomic data on a selection o f the actinorhodin genes also confirmed the findings, in S. coelicolor repression in the p p k m utant and activation in the phoP mutant, and conversely activation o f act genes in both mutants o f S. lividans, all in com parison to the parental strains.

In conclusion phosphate lim itation causes a whole cascade o f genes to be regulated in

Streptomyces, alongside a b rief period o f transition coinciding with a decrease in

biomass and the initiation o f actinorhodin synthesis. The tw o m utants give different results but show m ore obvious stress than the parental strains. p h o P mutants generally behave the same betw een the two species, how ever the inactivation o f p pk, induces actinorhodin synthesis in S. lividans, but represses production in S. coelicolor. PhoP has m ultiple targets induced at times o f phosphate limitation, including p p k either directly or indirectly.