As previously published, ph o P inactivation influences actinorhodin synthesis (Ghorbel
et al., 2006; Sola-Landa et al., 2003); the ph o P mutants o f both S. coelicolor and S. lividans, synthesised increasing titres o f actinorhodin relative to those present in
either o f the parental strains. In the S. coelicolor study, this synthesis coincided with phosphate exhaustion and the transcriptomic data gave additional confirmation. Across the time points, an initiation o f expression o f some o f the actinorhodin genes occurred between 31 h and 42 h, depending on the specific individual gene. Similarly the metabolic flux-related analysis confirmed that actVA 1, a ctV A l, actVA5 and actIORF3 were am ong the significant metabolic genes changing between the phoP (42 h) mutant and the wild type (38 h) (A.M. Kierzek, personal communication). In comparison the
p p k m utant showed a decreased actinorhodin synthesis, and from inspection o f the
transcriptomic data against the same act genes a decrease in transcription occurred from 31 h onwards.
A well know n system concerned w ith phosphate lim itation is the p s t operon; some members will be discussed alongside some additional prospective members found from the transcriptom ic analysis o f both studies. Comparing the data between the p p k and the
phoP m utants in S. coelicolor, there is a clear difference in the behaviour o f the two
com ponent system, pho R P and the p s t operon (pstSCAB). In m utants where the phoP has been inactivated, there is complete and significant down-regulation o f both phoR and phoP , in addition to pstC . Here the data between S. lividans and S. coelicolor differs
slightly; the S. lividans has tw o members o f the p s t operon significantly repressed, p stS and pstC , but neither p h o R or phoP are present, although both are down-regulated just outside the significance threshold. In S. coelicolor, p stC is present and significantly down-regulated, along w ith phoR and p h o P but not p s tS (w hich w ould be expected). W hen studying the rem aining members o f the p s t regulon, in both S. coelicolor and
S. lividans pstA and pstB are detected but not with significantly altered transcription.
Since both studies used the oligonucleotide arrays and neither pstA nor pstB appear in either significant gene list, perhaps the actual probes could be affected, maybe not having such efficient binding therefore producing low signal intensities but still viable. To clarify, other m ethods could be used, in fact the p s tS gene was subjected to northern blot analysis, and was shown to have the correct and similar profile to pstC which was dem onstrated to be down-regulated by the microarray data.
The transcriptional activator o f the p s t operon is the PhoP protein, and phoR and phoP were dow n-regulated in S. coelicolor, w hen compared to the wild type strain, confirming there is inactivation o f the phoP; this is consistent w ith PhoP functioning as an auto-activator o f its ow n expression and o f phoR . H ow ever in the S. lividans study, complete dow n-regulation o f either phoR or phoP did not occur. The phoR remained slightly repressed more so than phoP, but the results were not as statistically significant as the same system in S. coelicolor. Southern blots and antibiotic sensitivities verified the S. coelicolor m utants; perhaps further verifications need to be conducted on the
S. lividans m utants (which were constructed by another laboratory). However
combining the actinorhodin elevated synthesis, the phoP m utant is probably inactivated, but maybe there was only a partial inactivation. I f the raw values from the microarray experiments are taken into account (before normalisation) the values were relatively low similarly w ith S. coelicolor phoP m utant and few spots are deem ed ‘b ad ’ spots, most achieving a flag o f ‘A ’.
The m ost overly expressed gene o f the entire S. coelicolor study encoded a protease (SC 04157). Interestingly the next highest significant genes encode a two component system im m ediately dow nstream o f this protease (S C 04155 and SC 04156). These three genes were consistently proved to be over expressed in the pho P m utant irrespective o f w hich analysis m ethod was performed, present in both the GeneSpring and rank product
analysis, as well as the metabolic flux-related analysis. M oreover this expression o f the protease (S C 04 15 7) has been verified, using two further techniques: the northern blot and m utational studies. As part o f an undergraduate project, S. Forrest (personal com munication) generated a single mutant o f both the protease and the response regulator o f the two com ponent system in S. coelicolor M T1110 (Chapter 4.3.3). As illustrated by grow th studies and antibiotic analysis the knockout m utant o f the protease precociously hyperproduced undecylprodigiosin under phosphate limitation. The response regulator seemed not to initiate such an extreme reaction, a slight increase o f undecylprodigiosin occurred but not to the same extent as w ith the protease. The protease m utant produced a further peculiar result exhibiting a decrease in cell biomass and hence growth rate over time; this is a very prelim inary result since only two time points were recorded w ith no replicates, but both the phosphate limited and phosphate replete conditions produced the same result.
Since the response regulator (S C 04156) o f the two com ponent system seems to have a relatively m inor effect on antibiotic production, it possibly indicates that either PhoP negatively regulates the system, or that phoR P is the stronger system, dominating when both systems are present (as in the wild type) or if the S C 04 1 5 6 system is inactivated. H owever w ith the protease, the pathway is more com plicated, induced with the inactivation o f p h o P and affecting antibiotic synthesis in the S C 04157 mutant. Originally the protease was chosen for further study by using the original transcriptomic analysis generated from the S. coelicolor study o f the phoP m utant and wild type. The unexpected result o f the protease m utant (S C 04157) obtained from the initial growth curve studies suggests that this gene might play a role in the scavenging o f phosphate in phosphate starved cultures.
Protein BLA ST searches o f S C 04157 suggest this gene is a D egP/H trA homologue, being more com m only classified as degP in E. coli and htrA in B. subtilis. The protease works as quality control protein for other proteins, either as a chaperone or a protease. It can digest any badly m isfolded proteins in a protease capacity or by working as a chaperone assisting proteins that can be refolded and, if unsalvageable, reverting back to the protease function degrading the protein. Previous studies show that the expression o f
is activated, degrading (deg) m isfolded proteins (Jomaa et al., 2006). However at lower tem peratures the protein undertakes a chaperone function, aiding the correct folding o f newly generated proteins. These studies have been com pleted in E. coli and B. subtilis, but w ith no previous knowledge in Streptomyces, both functions could be occurring simultaneously, w ith both the protease and chaperone activity functioning. Even though a protease and m olecular chaperones perform antagonistic functions, it is thought they share com m on features since their substrates are very sim ilar (Spiess et al., 1999).
In other studies degP/htrA has been shown to be regulated by a two component system, encoded by cpxAR and cssRS in E. coli and B. subtilis, respectively (Raivio and Silhavy, 1999; D annon et al., 2002); no obvious highly homologous tw o-com ponent system has been discovered as o f yet in Streptomyces. Protein blast sequences o f SC 04155 (response regulator) and S C 04 1 5 6 (sensor kinase) showed m atches w ith both the cpxAR and cssRS, o f course m ainly the similarities will be due to the fact that two component systems are conserved w ithin and between species. H owever com bining those findings w ith the transcriptom ic data, showing an over expression o f a two com ponent system adjacent to the protease gene perhaps indicates all three com prise an operon. With the protease, degP/htrA pointing towards this two com ponent system (SC 04155 and S C 04156) being a hom ologue to either the CpxAR or the CssRS systems.
Since all the physiological growth studies were carried out at a specific temperature, and the function o f the protease has shown to be temperature dependent, this could alter the pathway or m ode in w hich this protease works. In E. coli, DegP functions as a protease, degrading m isfolded proteins above 28°C, and below that tem perature acting as a chaperone, aiding the folding o f proteins (Jomaa et al., 2006). The S. coelicolor study was grown at 30°C, suggesting the protease should be functioning as the protease, aiding the degradation o f misfolded periplasmic proteins. H ow ever the difference in temperature is slight (2°C) and performed in Streptomyces w ith a relatively unknown function, so the DegP could be functioning as both a chaperone and a protease. Obviously some internal imbalance is occurring, since the precocious antibiotic production suggests the cell is under stress, wrongly folded or undegraded proteins could cause such stress.
The CssRS system in B. subtilis and the CpxAR system o f E. coli bear at least some resemblance to each other w ith both being two com ponent regulatory systems. W hen the relevant response regulators and sensor kinases o f each respective pair have their protein sequences blasted, they show high amino acid similarity. Both systems are auto regulated and involved in controlling the transcriptional gene expression o f HtrA like proteases. (B. subtilis, htrA and htrB; E.coli, degP) (Darm on et al., 2002). However even though both show high similarities, the cssRS system prom oter regions do not appear to have the CpxRA consensus binding site (Raivio and Silhavy, 1999). In addition both systems respond to different stimuli, CssRS to heat shock and CpxAR to pH change or alterations in the inner membrane lipid com position (Raivio and Silhavy, 1999). The C pxA R system has been shown to regulate degP in conjunction with the sigma factor a E, the anti-sigm a factor RpsE and two phosphoprotein phosphatases, PrpA and PrpB (Pallen and Wren, 1997).The CssRA system o f B. subtilis causes the induction o f htrB (alongside htrA), w hen the protein sequence is blasted using the ScoDB database num erous genes were generated including S C 04157 (similar since they are both proteases), how ever the only other gene present in the up-regulated data list was S C 03977 (+0.6, pfp < 0.011).
DegP functions in the periplasm o f Gram-negative bacteria such as E. coli, in contrast Gram-positive bacteria such as B. subtilis lack an outer membrane wall. Instead comprising o f only one thick network o f cell wall polymers, and hence an absence o f a periplasm (H yyrylainen et a l , 2001). In B. subtilis upon translocation, secretory proteins fold into their natural confirm ation at the membrane cell wall interface, before being released into the environm ent (H yyrylainen et ah, 2001). The presence o f three paralogous genes (ykdA, yvtA and yyxA , encoding htrA/Do proteases) found in B.
subtilis suggests that as with the Gram-negative bacteria, m isfolded and surplus proteins
are rem oved from the cell wall membrane interface o f Gram-positive bacteria by proteases w ith a clean up function (Tjalsma et al., 2000). A possible pathway for the same system occurring in the Gram-positive Streptomyces.
Since the protease D egP/H trA is regulated by a two com ponent system in both E. coli and B. subtilis, and three genes (suspected DegP, S C 04157 and a two component system, S C 04156 and S C 04167) were massively up-regulated in the phoP m utant
transcriptom ic data, it suggests these genes could belong in an operon. Studies have been perform ed in E. coli and B. subtilis, providing pathways w hich could be used to deduce the actual role in Streptomyces. It is possibly more likely that the B. subtilis system is m ore relevant and closely related, since both organisms are Gram -positive, lacking the periplasm. In both E. coli and B. subtilis, the protease and the two com ponent system are positioned far away from each other, w hereas the proposed regulatory system o f the protease in Streptomyces is adjacent. H owever these are only prelim inary findings, m ore follow up work needs to be com pleted, but at present the results indicate that the protease and two com ponent system are in some way regulated by PhoP, w hether directly or indirectly. The protease alone increases antibiotic synthesis, although w hether this increased production is due to a general stress or specifically phosphate lim itation cannot be concluded. This system appears to be a prime candidate for a more targeted and detailed study, deducing the possibly pathway and link with phoP.
B oth the S. lividans and S. coelicolor phoP mutants have sim ilar genes being repressed or expressed, indicating they m ust be highly involved in the pho regulon and phosphate regulation. A ny sim ilarities w ould prove interesting since the two different strains (S.
lividans and S. coelicolor) were grown on two separate phosphate-lim ited media; one a
minimal chem ically defined media and the other a com plex media. The complex media included yeast extract and casam inoacids so in fact could have a higher concentration o f phosphate than originally stated (0.37 m M in R2YE com pared to 2 mM in Evans). These two experiments were carried out using very different methodologies, the com plex m edia was perform ed on solid m edia agar plates overlaid with cellophane and the m ycelium scraped, com pared against the minimal m edia grown using a pH controlled ferm enter in liquid culture. Due to the nature o f plate growth the internal com position o f each plate cannot be controlled possibly resulting in small microcosms o f habitats w here the aerobic and nutritional com positions can not be maintained. W hereas in the m inim al m edia only the bare m inimum o f reagents were added, allowing for a more detailed study o f metabolites and antibiotics to verify any effects discovered.
Other regulatory systems were found to change depending on the mutation. A whole range o f ribosom al proteins had reduced expression at 46 h, coinciding w ith the time o f
the transition phase, consistent with results published by Blanco et al., (2001). In
Streptomyces, this phase is associated w ith an increase in intracellular ppGpp, inducing
the stringent response (Strauch et al., 1991). In E. coli the changes in ppGpp and the stringent response initiates a down regulation o f rRNA and protein promoters as a response to starvation conditions (Cashel and Rudd, 1987). Another ribosomal elongation factor G, fu sA was also down-regulated in the phoP mutants, a GTPase involved in the translocation o f ribosomes along mRNA during protein biosynthesis (Rodnina et al., 1997), indicating that under phosphate lim itation or phoP inactivation, some protein synthesis is occurring, perhaps for genes involved in phosphate scavenging or related to cell stress.
M ost bacterial proteins are stable, how ever some w hich due to their important physiological roles are relatively unstable, and are broken down w ithin a few minutes (Gottesman and M aurizi, 1992). Generally maintenance and quality control o f proteins are perform ed by tw o classes o f protein, m olecular chaperones and ATP-dependent proteases, w ith Lon being a m em ber o f the latter class (Sobczyk et al., 2002). Lon, a protein involved in the degradation o f abnormal proteins (Tom oyasu et al., 2001) is present in the up-regulated genes o f the S. coelicolor phoP mutant. Conversely DnaK, a m olecular chaperone is dow n-regulated in the same study.
A nother protease likely to have quality control function for m em brane bound proteins is FtsH (S C 05587) (Tjalsm a et al., 2000). ft s H was present in the down-regulated genes o f the p p k m utant, encoding for a zinc binding metalloprotease. B. subtilis fts H mutants showed high sensitivity to heat and salt stresses, whereas in E. coli fts H has been shown to involved in membrane protein assembly and the degradation o f unstable proteins (Akiyama et al., 1996). In particular fts H has been shown to be the principal method o f SecY degradation, m utants in fts H show high levels o f SecY, detrimental to cell growth and protein export (K ihara et al., 1995). SecY alongside SecE are important in protein translocase o f E. coli, form ing a complex with SecG (Tokuda, 1994). Since there is down-regulation o f f t s H in the p p k mutant, it could suggest some kind o f stress mechanism not functioning as normal in the p p k mutant, possibly explaining the innate sickness o f the cell. Follow ing the B. subtilis model, the low level o f expression o ffts H in the p p k m utant leads to low levels o f the FtsH protein being encoded, therefore less
protein to perform the proteolytic degradation o f SecY, increasing levels o f the protein which has been show n by m utational studies to be detrimental to the cell.
ram S and ramB are both down-regulated in the p h o P m utant o f S. coelicolor, but only ram S in S. lividans, suggesting that the ram system m ight in some function be a PhoP
target, since the inactivation has caused ram S to be repressed too. The ram cluster is well classified consisting o f ramAB (ABC transporters), ram C (serine/threonine kinase),
ram S (encoding a small protein) and the response regulator, ramR (Keijser et al., 2002).
A lantibiotic, SapB, a small secreted peptide acting as a biological surfactant aiding aerial hyphae to cross the soil air barrier, is thought to depend on ram C and ram S for its production (ICodani et al., 2004). Null mutants in both these genes resulted in the typical bald phenotype, sim ilar to bid mutants where aerial hyphae are not formed (O'Connor and N odwell, 2005).
Since ram S is repressed in both S. coelicolor and S. lividans it is therefore not a media specific occurrence, as one was performed on a m inimal medium and the other complex. The initial finding o f ram S repression was discovered in the S. coelicolor study conducted in minimal liquid media, w ith no obvious need o f the SapB surfactant for aerial hyphae to cross the air surface barrier possibly explaining the repression. However the discovery o f the same phenom enon in S. lividans grown on plate cultures suggests that the m utation in phoP or phosphate lim itation is playing some functional role. Could ram S be acting as some secondary pathway? Aerial hyphae give rise to differentiation and eventually sporulation, under phosphate lim itation this process would need to be hastily perform ed for the protection o f viable spores until the phosphate lim itation has passed. The PhoRP system senses phosphate limitation, inducing a w hole cascade o f genes involved in scavenging phosphate and secondary metabolite production, if in addition it could induce the ram operon as secondary signal alerting the plight o f lowering phosphate levels, increasing aerial hyphae and SapB levels, allow ing for the passage through the soil and into the air, resulting in the eventual release o f the spores, hopefully avoiding desiccation and starvation. Aerial hyphae have been shown to coincide w ith the transition phase (Susstrunk et al., 1998) and the production o f antibiotics (Thom pson et al., 2002) both corresponding with decreased phosphate limitation and initiation o f PhoP activation.
A nother o f the genes in com m on between the two phoP mutants, was tesB (SC O l 153) encoding a acyl coA thioesterase type II, functioning as an alternative chain terminating enzyme tow ards m edium length acyl thiesterases (Kotowska et al., 2002). The thioesterase was up-regulated in both mutants, however in the S. coelicolor study an additional thioesterase, tesB2 (S C 02773) was up-regulated in S. lividans, scoT (S C 06287), hence all three thioesterases are up-regulated across the phoP mutants o f both species. Thioesterases form two distinct groups, thioesterases type I (such as TesA in E. coli) and thioesterases type II (TesB, ScoT), catalysing the hydrolysis o f saturated (3-hydroxy-acyl CoA, w ith TesB (Cg-Cis) being more universal in chain length than TesA (C i2-C 18) (Barnes et al., 1970). N ot m uch is known about the physiological role o f TesB in the cell, one function is to prevent high accumulations o f intra cellular acyl CoA due to the high specificity o f the enzyme for the com pound (Zheng et al., 2004).
3-Hydroxyalkanoic acids (3HA) are thought to maybe act as intermediates in the synthesis for m any chemicals, such as antibiotics, aromatics and pheromones. The m onomers could jo in forming polyhydroxyalkanoates (PHA), macromolecules synthesised w hen grow n on multiple carbon sources, functioning as reservoirs for carbon and reducing equivalents. In E. coli, it was found that TesB alongside PhaG, redirects the fatty acid biosynthesis to 3HA production as well as PH A synthesis (Zheng
et al., 2004). H ow ever in S. coelicolor thioesterases are involved in the same
condensation and reductions as shown w ith fatty acid biosynthesis, but are instead used in the generation o f polyketides, in fact mutants in TE II have a reduced synthesis o f polyketide production by 90% or more (Xue et al., 1998; Butler et al., 1999). In both the S. coelicolor and S. lividans studies the ph oP mutants resulted in an over production o f actinorhodin, a well know n polyketide (Crump et al., 1996) w hen compared to their respective w ild types. Therefore the induction o f the type II thioesterases could be a reflection o f this increased synthesis o f the secondary m etabolite.
Some o f the m ost interesting dow n-regulated genes generated by both studies in the
phoP mutants have relatively unknow n functions, for exam ple SCO 1048, encodes a
secreted protein but not even perform ing a BLA ST search against this can suggest a related function in another organism. Another two interesting genes, S C O l968 and