Cyanosite webserver: http://bilbo.bio.purdue.edu/www-cyanosite/

Cyanobacterial Research

I. Background and Research from 1993 to 2003

A. Research Summary and Background
B. Nitrogen Fixation in Cyanothece
C. Synechocystis Mutations and a Structural Model of MSP
D. Control of Photosynthesis in Cyanothece so. ATCC 51142
E. Impact of Iron Deficiency on CHL-protein Complexes
F. Impact of Altered Fe and Nitrogen Concentration on Photosynthesis
G. Differential Gene Expression
H. Construction of a full-genome cDNA microarray for Synechocystis
I. Redox Regulation of photosynthesis via analysis of Synechocystis mutants
J. Microarray Analysis of the Genome Wide Response to Iron
Deficiency and Iron Reconstruction K. Bibliography

II. Gene Regulation and Environmental Stress

A. Oxidative Stress
The Synechocystis microrrays provided a great deal of information on the impact of oxidative stress on this organism. We determined that gene slr1738 encoded a protein similar to PerR in Bacillus subtilis and was induced significantly by peroxide. We constructed a PerR knockout strain and use it to help identify components of the PerR regulon. PerR was next to sll1621 which codes for a peroxiredoxin and share a divergent promoter that is regulated by PerR. We also found that isiA was strongly upregulated by hydrogen peroxide. In addition, slr1894 encoded a protein similar to MrgA in B. subtilis and was also upregulated by peroxide. In addition, we determined that a ?MrgA knockout mutation was highly sensitive to peroxide. These results are detailed in publications Li et al.(2004) and Singh et al. (2004).

B. Insights into Regulation of isiA
The function of IsiA under different environmental conditions has remained an enigma. During this past period, we analyzed the regulation of IsiA under normal growth conditions, as well as under stress conditions in a ?isiA mutant. In Singh et al. (2005), we monitored the presence of IsiA by four different procedures—absorbance at 673 nm, authentic IsiA-Chl complexes, the presence of the protein as detected by antibody and by CAT activity (generated by the isiA promoter in the CAT pSB2A plasmid). Under typical batch growth conditions at 40 ?mole photons m-2s-1, we show that the isiA promoter attached to CAT peaked at 6 days, as did the absorption peak; the IsiA protein itself was maximal at about 7 days. Interestingly, IsiA complexes in green gels were visible as early as 3 days and reached a high level by 4 days. These results emphasize the many steps involved in synthesizing, assembling and inserting a Chl-protein complex into the membrane and the key role that IsiA can play in understanding these phenomena.

The complex dynamics of the CAT activity is shown in Fig. 3 of Singh & Sherman (2006). The dynamic regulation of isiA during the long-term growth is remarkable and quite different than that described in less-detailed reports. Interestingly, the addition of peroxide had very little impact on CAT activity and this was a case where there was significant accumulation of transcript, but no detectable IsiA protein. In total, these results indicated that there is some post-transcriptional event(s) that determines whether IsiA is produced and integrated into a stable IsiA-Chl complex. Interestingly, transcription of the slr0513 gene and psbA2 followed similar patterns to that of IsiA. Such results suggest that IsiA accumulation during this light-limited, stationary phase may be linked to PSII protection. It is becoming obvious that IsiA can be transcribed under a variety of different growth conditions—some stress, some normal—but that various post-transcriptional mechanisms will determine how IsiA is incorporated into the membrane and, possibly, whether or not it is involved with PSII or PSI.

The results with the ?isiA mutant also provided some surprising results (Singh et al. 2005; Singh & Sherman 2007). We determined that one of the main differences between ?isiA and the wild type under normal conditions was the induction of a gene cluster (sll1693-sll1696) that encoded genes resembling pilins or general secretory proteins (Gsp). These proteins are targeted to the cytoplasmic membrane and we suggest that they may be involved in the assembly of membrane complexes including pigment-protein complexes. In addition, ?isiA is more resistant to peroxide compared to the wild type. Importantly, in the presence of peroxide a cluster of genes that includes a peroxiredoxin was induced 7-8-fold and we suggest that this peroxide-scavenging enzyme is responsible for the increased peroxide resistance of the ?isiA strain.

The microarray analysis of differential gene expression in ?isiA has identified some important gene clusters (Singh et al. 2005; Singh & Sherman 2007). We summarize the various hypotheses for IsiA function in Singh & Sherman (2007) and conclude that it is very likely that IsiA is involved in a number of ways in the assembly and protection of photosystem complexes. We also suggest that IsiA may also be involved with the cytoplasmic membrane since one of the key features of the ?isiA mutant is an enhancement of transcription for genes encoding proteins destined for the cytoplasmic membrane and the periplasm. Additionally, we speculate that IsiA may be involved with the transfer of electrons to extracellular electron acceptors. The inductions of the pilins occurred whenever PSI or IsiA were absent, conditions that can lead to charge accumulation due to somewhat slow or faulty electron transport. This may place an emphasis on charge dissipation to the exterior and require the synthesis of additional pilins that may for extracellular nanowires which can transfer electrons from the cell surface to some external compound. Such nanowires have been observed in Synechocystis and this finding opens up an entire new avenue of research relating to photosynthetic electron transport and photosynthetic microbes.

C. MrgA—Oxidative Stress and Fe Storage
An interesting current project concerns the MrgA protein. We first identified this protein in our oxidative stress experiment and found this to be part of the PerR regulon. We are currently studying this protein from two directions. First, in a ?mrgA mutant, among the gene changes that occurred in the mutant relative to the wild-type, there were two major gene clusters that appear to be involved with the biosynthesis of exopolysaccharides (EPS) and other proteins destined for the cell wall. Thus, we began studying the relationship between oxidative stress damage and the EPS. In order to do so, we have needed to develop some new techniques, including the study of various stains via fluorescence microscopy. These experiments are being done in connection with Dr. Jamie Foster, a former postdoc in the lab. Our working hypothesis is that ?mrgA cells are less sensitive to peroxide killing as the culture approaches stationary phase than while growing exponentially due to the large increase in EPS that occurs during growth. To test this hypothesis, we have knocked out one of the gene clusters involved with extracellular EPS production and are using that in our studies. Our preliminary data supports our working hypothesis. We have provided the ?mrgA strain, as well as the above mutant strains, to Dr. Nir Keren of the Hebrew University in Israel. He had been studying Fe storage in the bacterioferritin proteins in Synechocystis. However, the deletion of both Bfr genes indicated that other proteins were obviously involved and we decided to investigate the importance of MrgA as an Fe-storage protein. Work done in both laboratories indicates that ?mrgA cultures grow less well in a Fe-deplete medium, and experiments indicated that there is an impeded utilization of the intracellular iron. Our current results suggest that MrgA plays an important role in the transport of intracellular Fe fromstorage in the bacterioferritins, to biosynthesis of metal cofactors during cell growth.

At the current time we have two hypotheses for the overall role of MrgA in Synechocystis.  First, it appears to be a critical Fe storage/transfer protein that likely interacts with the bacterioferritins in order to make Fe bioavailable to the cell.  In addition, the lack of MrgA in the cell appears to once again affect the external surface of the cell.  More exopolysaccharide is produced and excreted and, with time, this helps cells become less susceptible to peroxide stress.

D. Models of Regulation During Light-Dark Transitions and of a Sigma Factor Network
We have devoted a great deal of effort to understanding the way in which the group 2 sigma factors function in Synechocystis, and especially during the light and dark periods.  The results have been published in a series of papers Singh et al. (2006), Foster et al. (2007) and Summerfield and Sherman (2007).  This has provided a great deal of information and much of this has been summarized in models presented in Summerfield and Sherman (2007).

Our results indicate the importance of the group 2 sigma factors, SigB, SigD and SigE, in global regulation of transcription in Synechocystis sp. PCC6803.  But, why are so many genes affected when one of these factors is deleted?  The results can best be explained using the σ cycle paradigm and particularly the stochastic release model as described in Mooney et al. (2005).  In this model, a pool of σ factors competes for binding to the core RNA polymerase to form an open complex.  The stochastic release model states that the affinity of σ for the RNA polymerase decreases in the elongation complex, but that release of the σ factor occurs stochastically after the RNA polymerase has initiated transcription.  Thus, sigma release occurs during each transcription cycle and sigma competition for rebinding of the RNA polymerase is permitted.  Mooney et al. (2005) have summarized the evidence for and against a half dozen different possible models of sigma action and conclude that stochastic release best describes all of the data.  Therefore, control of global transcription will be based on the amount of the various sigmas present and able to bind to the RNA polymerase.

The model described in Figure 2 is based on our transcription data as well as the levels of the nine sigma factors in the light and dark as calculated by Imamura et al. (2003).  In the wild type, the transition from growth in the light to the dark generated an increase in SigB and a decrease in SigE.  The major impact of this transition on global regulation was a decrease of the transcript levels of genes encoding ribosomal proteins and photosynthesis proteins.  At the same time, genes encoding enzymes involved in energy metabolism, biosynthesis of various cofactors and prosthetic groups, regulatory functions, and transport and binding proteins tended to increase.  This pattern was reversed as the cells go back into the light where SigB decreases two-fold SigD and SigE increase two-fold.  When SigB is absent, there’s very little effect in the light.  However, in the dark, many more genes show enhanced transcript levels relative to the wild type, especially in genes encoding ribosomal proteins and many hypothetical proteins.  It is striking that the major effect of removing SigB is to up-regulate at least some genes in many different categories.  Thus, the absence of SigB in the dark permits other sigma factors to preferentially bind to the RNA polymerase and to transcribe selected genes—especially the major operon encoding ribosomal proteins.

The absence of SigD has a more profound impact on global regulation of transcription.  SigD represented a larger proportion of sigma factors available in both the light and the dark, but especially during light growth.  In accordance with the stochastic release model, our results clearly demonstrated a much greater influence of SigD removal in the light than in the dark.  In the light, many genes demonstrated differential transcription in DsigD compared to the wild type and many of them were up-regulated.  When SigD was absent, photosynthesis genes demonstrated numerous changes, especially in the light where the SigD concentration would be the highest.  The absence of SigD leads to an increase in approximately two-thirds of the differentially regulated genes in the light, whereas only approximately half of the genes are up-regulated in the dark.  Taken together, the result with both mutants indicate that SigB is much more important in the dark, whereas SigD has a much greater influence on global transcription in the light.

E.      Growth-phase dependent gene expression—light limitation and stationary phase.
One specific objective was to determine the nature of gene expression as the cells alter their physiological growth state in response to changes in light intensity and their nutritional and physical environment.  Under typical laboratory batch growth conditions, cyanobacteria grow exponentially then transition to a light limited stage of linear growth before finally reaching a non-growth stationary phase.  Our results indicated that there is widespread differential gene regulation during the change from exponential to linear growth in the cyanobacterium and that much of this is due to changes in light intensity based on self-shading Foster et al. (2007).  Specifically: 1) many photosynthesis and regulatory genes have lower transcript levels; 2) individual genes such as sigH, phrA, isiA which encode a group 4 sigma factor, a DNA photolyase and a chl-binding protein, respectively, were strongly induced; 3) the loss of SigB significantly impacted the differential expression of genes; this included the up-regulation of the photosynthesis and specific regulatory genes as well as a general down-regulation of the hypothetical and unknown genes.  This helped to develop the model in Fig. 2, and also gave important information on the sigma cascade, which led to the production of sigH to be used in transcription during the stationary phase and the importance of the photolyase phrA under these conditions.  In addition, we also determined that a cluster of genes sll1722 to sll1725 were strongly upregulated during the light limited phase.  These are genes that demonstrated homology to E. coli genes involved with exopolysaccharide production.  The results indicate that EPS production is enhanced in stationary phase and correlates with data from the DmrgA mutant that growth-phased dependent EPD production protected against peroxide stress.

F. Transcriptional reposnse to change in pH from 7.5 to 10

Many cyanobacterial strains are able to grow at a pH range from neutral to pH 10-11.  Such alkaline conditions favor cyanobacterial growth (e.g., bloom formation) and cyanobacteria must have developed strategies to adjust to changes in CO₂ concentration and ion availability. Synechocystis sp. strain PCC 6803 exhibits similar photoautotrophic growth characteristics at pH 10 and pH 7.5 and we examined global gene expression following transfer from pH 7.5 to pH 10 to determine cellular adaptations at elevated pH.  The strategies used to develop homeostasis at alkaline pH had elements similar to many bacteria, as well as components unique to phototrophic microbes.  Some of the response mechanisms previously identified in other bacteria included up-regulation of Na+/H+ antiporters, deaminases and ATP synthase.  In addition, up-regulated genes encoded transporters with the potential to contribute to osmotic, pH and ion homeostasis; e.g., a water channel protein, a large-conductance mechanosensitive channel, a putative anion efflux transporter, a hexose/proton symporter and ABC transporters of unidentified substrates.  Transcriptional changes specific to photosynthetic microbes centered on NADH dehydrogenases and CO₂ fixation.  The pH transition alters the CO₂/HCO3- ratio within the cell and the up-regulation of three inducible bicarbonate transporters (BCT1, SbtA and NDH-1S) likely reflects a response to this perturbed ratio.  Consistent with this was increased transcript abundance of genes encoding carboxysome structural proteins and carbonic anhydrase.

III. Membrane Biology Grand Challenge – with Dr. Himadri Pakrasi, Washington University and members of the EMSL lab at the Pacific Northwest National Laboratories