Project description:NtcA regulates primary in a unicelluar cyanobacterium Synechocystis sp. PCC6803. We constructed an ntcA-overexpressing strain, named NOX10, by introducing the ntcA genes fusing psbAII promoter by homologous recombination. The transcript profiles of parental wild-type strain GT and NOX10 were compared by microarray CyanoChip (Takrara bio.). Experiments were performed two times with biologically independent RNA. Total RNAs from two independent wild-type (GT) and two independent ntcA-overexpressing strains were differently labeled by Cy3 or Cy5, followed by hybridization on CyanoChip.
Project description:Rre37 activates sugar catabolism in a unicelluar cyanobacterium Synechocystis sp. PCC6803. We constructed rre37-overexpressing strain, named ROX370, by introducing the rre37 genes fusing psbAII promoter by homologous recombination. The transcript profiles of parental wild-type strain GT and ROX370 were compared by microarray CyanoChip (Takrara bio.). Experiments were performed by three times with biologically independent RNA. The results showed that genes for the oxidaditive pentose phosphate pathway and glycogen catabolism were induced by rre37 overexpression. Total RNAs from three independent wild-type (GT) and three independent rre37-overexpression strains were differently labeled by Cy3 or Cy5, followed by hybridyzation on CyanoChip.
Project description:NtcA regulates primary in a unicelluar cyanobacterium Synechocystis sp. PCC6803. We constructed an ntcA-overexpressing strain, named NOX10, by introducing the ntcA genes fusing psbAII promoter by homologous recombination. The transcript profiles of parental wild-type strain GT and NOX10 were compared by microarray CyanoChip (Takrara bio.). Experiments were performed three times with biologically independent RNA. Total RNAs from three independent wild-type (GT) and three independent ntcA-overexpressing strains were differently labeled by Cy3 or Cy5, followed by hybridization on CyanoChip.
Project description:SigE is a global regulator for sugar catabolism in a unicelluar cyanobacterium Synechocystis sp. PCC6803. We constructed SigE-overexpressing strain, named GOX50, by introducing the sigE genes fusing psbAII promoter by homologous recombination. The transcript profiles of parental wild-type strain GT and GOX50 were compared by microarray CyanoChip (Takrara bio.). Experiments were performed by three times with biologically independent RNA. The results showed that genes for the oxidaditive pentose phosphate pathway and glycogen catabolism were induced by SigE overexpression. Total RNAs from three independent wild-type (GT) and three independent SigE-overexpression strains were differently labeled by Cy3 or Cy5, followed by hybridyzation on CyanoChip.
Project description:HoxH is a subunit of hydrogenase in a unicelluar cyanobacterium Synechocystis sp. PCC6803. We constructed hoxH mutant strain, which the mRNA levels of hoxH reduced to 50% of the wild-type. Total RNAs from three independent wilde-type (GT) and three independent hoxH mutant were labeled by Cy3 followed by hybridyzation on a microarry chip generated by Agilent Technologies (Design ID 036279). Microarray is designed by Prof. Tomohisa Hasunuma, Kobe University.
Project description:Protein phosphorylation via serine/threonine protein kinases (Spk) is a widespread mechanism to adjust cellular processes toward changing environmental conditions. To study their role(s) in cyanobacteria, we established a collection of 11 completely segregated spk mutants among the 12 annotated Spk’s in the model cyanobacterium Synechocystis sp. PCC 6803. Screening of the mutant collection revealed that especially the mutant defective in SpkB encoded by slr1697 showed clear deviations compared to wild type (WT) regarding carbon metabolism, i.e., a reduced growth rate at low CO2 and in the presence of glucose, different glycogen accumulation patterns, and a higher tolerance to external H2O2 than the WT. The proteome of ∆spkB showed several distinct differences compared to WT, which indicate changes of the cell surface but also metabolic functions. A phospho-proteome analysis revealed decreased phosphorylation of the carboxysome-associated protein CcmM and the regulatory PII protein in the mutant compared to WT, whereas the allophycocyanin alpha subunit was stronger phosphorylated. The decreased phosphorylation of PII was verified in Western-blot experiments, indicating a clearly delayed PII phosphorylation in cells shifted from nitrate-containing to nitrate-free medium. Furthermore, the mutant ∆spkB showed differences in the state transition consistent with the changed phosphorylation of allophycocyanin. Collectively our results indicate that SpkB is an important regulator under different environmental conditions in Synechocystis and seems to interact in the PII phosphorylation and probably with further substrates in a kinase network.
Project description:We employed chromatin pull-down and deep sequencing to globally identify HetR DNA targets in vivo at 6 hours after fixed-nitrogen deprivation. We identified novel DNA binding targets of tagged HetR-6xHis and defined a consensus HetR binding site from these HetR target sequences. Chromatin pull down of hetR mutant strain UHM103 carrying pAM4375, which expresses HetR-6xHis, and a wild-type control. One dataset was collected.
Project description:Populus x canescens plants were grown under different nitrogen supply conditions. They were treated with NH4+ or NO3- based nitrogen sources in the concentrations of 0.4, 2.0 or 8.0 mM. Leaves were harvested after 3 weeks of treatment.
Project description:The 6S RNA is a global transcriptional riboregulator, which is exceptionally widespread among most bacterial phyla. While its role is already well-characterized in heterotrophic bacteria, we subjected a cyanobacterial homolog to functional analysis, thereby extending the scope of 6S RNA action to the special challenges of photoautotrophic lifestyles. This study reveals 6S RNA as an integral part of the cellular response of Synechocystis sp. PCC 6803 to changing nitrogen availability. Physiological characterization of a 6S RNA deletion strain (ÎssaA) demonstrates a delay in the recovery from nitrogen starvation. Significantly decelerated phycobilisome reassembly and glycogen degradation is accompanied with reduced photosynthetic activity compared to the wild type. Transcriptome profiling further revealed that predominantly genes encoding components of both photosystems, ATP synthase and the phycobilisomes were negatively affected in the ÎssaA mutant. In vivo pull-down studies of the RNA polymerase complex further indicate a promoting effect of 6S RNA on the recruitment of the cyanobacterial housekeeping sigma factor SigA, concurrently supporting dissociation of group II sigma factors during recovery from nitrogen starvation. According to these results, 6S RNA supports a rapid adaptation to changing nitrogen conditions by regulating the switch from group II sigma factors SigB / SigC to SigE / SigA dependent transcription. We performed microarray analysis of total RNA from wild-type and âssaA cultures that were starved for nitrogen for seven days and recovered over a period of 48 hours. Sampling time points were t1 = 1h +N, t2 = 4h +N and t3 = 22h +N after nitrogen recovery. Samples were taken in biological replicates.
Project description:Global gene expression of Synechocystis sp. PCC 6803 encapsulated in silica gel was examined by microarray analysis. Cultures were encapsulated in gels derived from aqueous precursors and gels derived from alkoxide precursors and incubated under constant light for 24 hours prior to RNA extraction. Cultures suspended in liquid media were also exposed to 500 mM salt stress and incubated under identical conditions, for comparison purposes. The expression of 414 genes was significantly altered by encapsulation in aqueous-derived gels (fold change >/= 1.5 and P-value < 0.01), the expression of 1143 genes were significantly altered by encapsulation in alkoxide derived gels, and only 243 genes were common to both encapsulation chemistries. Additional qRT-PCR analyses of four select genes; ggpS, cpcG2, slr5055, and sll5057, confirmed microarray results. These results illustrate that encapsulation stress is quite different than salt stress in terms of gene expression response. Furthermore, a number of hypothetical and unknown proteins associated with encapsulation and alcohol stress have been identified, with implications for improving encapsulation protocols and rationally engineering microorganisms for direct biofuel production. 16 samples; 4 biological replicates each of 4 treatments