Overview
The experimental growth data for each strain under Pi replete and Pi deplete cultures is shown in Figure 1. Logistic curve fitting and statistical analysis of the experimental growth data reveals no significant differences between the growth rates between Pi replete and Pi deplete cultures, with the exception of MIT9312 growth rates whereby Pi replete growth was significantly greater than Pi deplete growth (p < 0.05), as can be seen in Figure 1. It is important to consider the physiological status of the cells at the harvest point when considering protein relative abundances. Importantly, growth analysis shows that both MIT9312 and SS120 were in late exponential/early stationary phase at harvest, whilst NATL2A was in mid exponential phase. As the point of harvest differs for NATL2A, it would be difficult to directly compare the protein complement of NATL2A cells to either MIT9312 or SS120. Given this, the results for NATL2A will be discussed separately.
Thirty eight, 63 and 34 proteins were identified with 2 or more peptides for strains MIT9312, NATL2A and SS120 respectively (Additional file 1: Table S1) with no false positives. An overview of the respective proteomes, through plotting theoretical values of isoelectric points (pI) against molecular weights (MW) reveal significant bias towards low pI values (Additional file 2: Figure S1), with no further correlation to MW, relative protein abundance, nor total peptide hits per protein (data not shown). This bias may be an artefact of the mass spectrometric analysis, where peptides are protonated directly before entry into the MS in order to assist flight and detection. As a consequence, naturally occurring proton-donor peptides may be preferentially selected. However, as there are no observable correlations between pI and peptide hits per protein, we can be confident that the intracellular protein abundances reported are directly reflective of the physiological status of the cells. Indeed, when interrogating the proportion of proteins with ≥ 50% of peptide hits, we see similarities between strains, such as the presence of RplL, RbcL and CsoS1 (Additional file 1: Table S1), however all three proteins have pI values < 7. Nevertheless, a high pI protein, PetH, is present in both MIT 9312 and SS120 samples. Also, identified proteins from all three strains are located evenly across the genomes, and are representative of most major functional groups such as central metabolism, photosynthesis, transcription and translation, biosynthesis and nutrient acquisition (Figure 2A). Of the 105 unique proteins identified, 6 were found in all three strains (Figure 2B). They are the ATP synthase subunits AtpA and AtpD, the PSII protein PsbO, the nitrogen regulatory protein GlnK, rubisco subunit RbcL, and the carboxysome shell protein CsoS1.
Using relative abundance cut-offs of 1.6 and 0.6 fold differences to represent increased or decreased relative abundances [21, 22], 4 proteins were more abundant in MIT9312 and 4 were less abundant than the replete cultures. Within NATL2A, 6 proteins were more abundant and 1 was less abundant than the replete cultures. In SS120, 4 were more abundant and none were lower than the replete cultures (Figure 2A).
Nutrient acquisition
What is immediately apparent from our results is the differential abundance of Pi acquisition proteins exhibited by all three strains to being grown in 10 μM Pi. MIT 9312 demonstrates the greatest sensitivity to Pi-deplete media, whereby the Pi stress related porinPhoE is > 15-fold more abundant (Figure 3), the putative alkaline phosphatase PhoA appears to be > 9-fold greater, and the periplasmic Pi binding protein PstS > 3 times more than the replete cultures. This result is directly in line with an earlier proteomic assay of P stress in a HL ecotype, MED4 [21], and closely reflective of microarray analyses of both MED4 and MIT9313 [14], Synechococcucs WH8102 [23], measured alkaline phosphatase activity of MIT9312 [15] and in line with observed responses within earlier Pi depletion studies of other cyanobacteria [15, 24–26].
Within NATL2A, PstS abundance is significantly greater within Pi-deplete conditions, though with greater uncertainty (Additional file 1: Table S1). However neither PhoA nor PhoE was observed with mass spectrometry here, which is surprising as we showed previously that both PhoA and PhoE are greater in abundance alongside PstS in the high light ecotype MED4 [21], as is true with MIT9312 in this study. However, considering that NATL2A cells are in mid-exponential phase as opposed to early stationary phase this may indicate a progressive strategy of protein expression within the cells, however more work is needed to clarify this.
What was also unexpected, was the absence of any Pi acquisition mechanisms (as reflected in observed peptide identifications) within SS120 cells (Additional file 1: Table S1), allied with no significant difference in growth rates between Pi-replete and Pi-deplete cultures (p > 0.05). SS120 is deficient in most Pi acquisition genes [14, 15], however it does have two copies of PstS, neither of which were present in our assay. At first glance, this result appears counter-intuitive, as a 'very' LL strain typically present in vivo within Pi-replete environments would be expected to be adversely affected by a substantial decrease in Pi. However, the absence of a phoBRregulon suggests that the strain is incapable of regulating a response to shifts in environmental concentrations of Pi that are not immediately starvation inducing [27]. Curiously, this also infers that activation of the phoBR response mechanisms within MIT9312 and NATL2A were directly due to the mechanism's innate sensitivity to changing external Pi concentrations. This suggests that the intensity of response is directly proportional to external Pi concentration, coincidentally specific to each strain, and may be reflective of each strain's environmental niche and/or obligate cellular requirements.
Photosynthesis, biosynthesis and central metabolism
The exposure of all three strains to lower Pi concentrations appears to have had little effect upon the photosynthetic machinery (Figure 4A and Additional file 1: Table S1). This is unusual, as Pi depleted conditions have been previously noted to directly affect both photosystems in cyanobacteria [21, 23, 28]. In contrast, it is interesting to note that, for MIT9312, both Rubisco subunits (RbcL and RbcS) are noticeably lower in abundance (Figure 3B). This suggests that there is a progressive strategy within the cell when acclimating to lowered Pi, whereby photosynthesis is initially dissociated from glycolysis, to then strategically break down the photosynthetic apparatus. This is a reasonable conclusion, considering a Pi-induced organised break down of phycobilisomes has been previously observed in Synechococcus sp. PCC 7942 [29], chlorosis has been observed in thermophillicSynechococcus under Pi-stress [28], and a strategic approach to a reduction in photosynthetic function has been hypothesised in MED4 [21]. Indeed, within WH8102 it appears that PSII was degraded before PSI, allowing continued cyclic photophosphorylation-based ATP generation to continue [23]. In this context, this could explain why an essential chlorophyll biosynthetic protein (ChlP) appears to be less abundant within Pi-deplete MIT9312 cells (Figure 4B). However, it would be parsimonious to also expect a concurrent reduction in the light harvesting protein (Pcb) within Pi-deplete MIT9312, which was noticed in MED4 [21], but there is no change. The reason for this is not clear.
When considering NATL2A solely, there appear to be a few subtle discrepancies in protein abundances between stressed and non-stressed cultures. Fumerase (FumC) is an enzyme associated with both the tricarboxylic acid (TCA) cycle and arginine/proline biosynthesis, and appears to be more abundant within NATL2A cells when Pi-deplete (Additional file 1: Table S1). As NATL2A has an incomplete TCA cycle, it is safe to assume that its function within the cell is within arginine and proline metabolism. Also, the acyl carrier protein (AcpP) is an essential component of fatty acid biosynthesis, and is more abundant in Pi-deplete NATL2A cells (Additional file 1: Table S1). Fatty acids are for the most part used within either fuel storage or membrane manufacture. However it may be misleading to arrive at the conclusion that this is a specific cellular response to lower Pi concentrations. It is possibly a function of apparently slightly elevated (albeit not significant) growth within NATL2A Pi-deplete cultures, and as such could reflect comparatively greater metabolic activity. Nevertheless, this explanation cannot immediately address the lower abundance of CobJ, a Precorrin-3B C17-methyltransferase region-containing protein (Additional file 1: Table S1), part of the aerobic vitamin B12 biosynthesis pathway within Pi-stressed cells. However, B12 synthesis is a sub pathway offshoot from the main chlorophyll biosynthetic pathway, and as such may reflect a metabolic preference for chlorophyll production that, again, may be representative of faster growing populations.
An interesting observation is the abundance of CitT within Pi-stressed SS120 cells (Figure 3A). This protein functions as a di/tricarboxylate transporter, which implies that the cells are scavenging lysed cellular material from the environment. That stressed SS120 cells appear to be preferentially acquiring tricarboxylic acid intermediates when growing in Pi-deplete conditions, and not upregulatingPstS, is puzzling. However, it may indicate that this strain may be supplementing an affected glycolysis pathway through acquiring external carbon sources, and that this is more evidence that the cells response to an environmental stress is an iterative, evolving process. SS120 may simply have not initiated transcription of PstS in sufficiently detectable quantities. Indeed, even in starvation experiments pstSexperession is far from an immediate response [14, 23].
Other proteins
An interesting observation is the presence of LuxR, the response regulatory family protein involved in quorum sensing within bacteria, in NATL2A cells (Additional file 1: Table S1). To our knowledge, this is the first instance of observing proteins putatively indicated in quorum sensing capability in any marine cyanobacteria. However, we were unable to locate any LuxI homologues, an essential protein required for effective quorum sensing, within NATL2A (data not shown). However LuxR is known to be a transcriptional regulator activated when cell concentrations of a particular trigger compound (usually N-(3-oxohexanoyl)-L-homoserine lactone, which is generated through the enzymatic functioning of LuxI) reach particular levels. As such, we speculate that the protein acts as a density-dependant transcriptional regulator, but for an unknown function, and through another trigger compound.