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 ΔisiA 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 μmole 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 ΔisiA mutant also provided some surprising results (Singh et al. 2005; Singh & Sherman 2007). We determined that one of the main differences between ΔisiA 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, ΔisiA 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 ΔisiA strain.
The microarray analysis of differential gene expression in ΔisiA 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 ΔisiA 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.
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