Characterization of the subdomains in the N-terminal region of histidine kinase Hik33 in the cyanobacterium Synechocystis sp. PCC 6803

Histidine kinase Hik33 responds to a variety of stress conditions and regulates the expression of stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. However, the mechanisms of response and regulation remain unknown. Generally, a histidine kinase perceives a specific signal via its N-terminal region. Hik33 has two transmembrane helices, a periplasmic loop, and HAMP and PAS domains in its N-terminal region, all of which might be involved in signal perception. To investigate the functions of these subdomains in vivo, we expressed a chimeric histidine kinase (Hik33n-SphSc) by fusing the N-terminal region of Hik33 with the C-terminal region of a sensory histidine kinase that is activated under phosphate-deficient conditions, SphS. Hik33n-SphSc responded to several stimuli that are perceived by intact Hik33 and regulated expression of the phoA gene for alkaline phosphatase, which is normally regulated under phosphate-deficient conditions by SphS. We introduced genes for modified versions of Hik33n-SphSc into Synechocystis and monitored expression of phoA under standard and stress conditions. Hik33n-SphSc lacking either the transmembrane helices or both the HAMP and PAS domains had no kinase activity, whereas Hik33n-SphSc lacking the HAMP or the PAS domain enhanced expression of phoA. Moreover, variants of Hik33n-SphSc, in which the membrane-localizing region was replaced by those of other histidine kinases, also responded to stress conditions. Thus, transmembrane helices, regardless of sequence, appear to be essential for the function of Hik33, while the HAMP and PAS domains play important roles in regulating kinase activity in vivo.     

Phosphate sensing in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803: SphU and the SphS–SphR two-component regulatory system

The Pho regulon is controlled by the histidine kinase-response regulator pair SphS–SphR in many cyanobacteria and up-regulation of the Pho regulon can be monitored by measuring alkaline phosphatase activity. However, the mechanism regulating signal transduction between SphS and SphR has not been described. We have created a cyanobacterial strain allowing the introduction of mutations into the transmitter domain of SphS. Mutations at Thr-167, adjacent to the H motif of SphS, introduce elevated alkaline phosphatase activity in the presence of phosphate and an enhancement of alkaline phosphatase activity, when compared to the control strain, in phosphate-limiting media. SphU acts as a negative regulator of the SphS–SphR system in Synechocystis sp. PCC 6803 and we show that constitutive alkaline phosphatase activity in the absence of SphU requires signal transduction through SphS and SphR. However, constitutive activity in the absence of SphU is severely attenuated in the ΔSphU:SphS-T167N mutant. Our data suggest that Thr-167 contributes to the mechanism underlying regulation by SphU. We have also assembled a deletion mutant system allowing the introduction of mutations into SphR and show that Gly-225 and Trp-236, which are both conserved in SphR from cyanobacteria, are essential for activation of the Pho regulon under phosphate-limiting conditions.     

The role of three-tandem Pho Boxes in the control of the C-P lyase operon in a thermophilic cyanobacterium

Cyanobacteria have an inherited advantage in phosphonate phytoremediation. However, studies on phosphonate metabolism in cyanobacteria are rare and mostly focus on physiology and ecology. Here, C-P lyase gene cluster regulation in an undomesticated thermophilic Synechococcus OS-B′ was examined in Synechocystis sp. PCC6803, a unicellular cyanobacterial model. Phylogenetic and cluster synteny analysis of C-P lyase genes revealed a closer relationship between Syn OS-B′ and Thermus thermophilus, than with other cyanobacteria. Pho boxes were identified in the 5′-end-flanking region of the C-P lyase gene cluster, through which the downstream gene expression was regulated in a phosphate concentration-dependent manner. Unexpectedly, the phosphate concentration that thoroughly inhibited Pho boxes was almost two orders of magnitude higher than that of any natural or anthropogenic wastewater reported so far. The Pho boxes mediated regulation was achieved through the Pho regulon two-component system, and the absence of either SphS or SphR ablated the cell's ability to sense ambient phosphate changes. The three tandems of Pho boxes maintained inequivalent roles, of which the third tandem was not essential; however, it played a role in adjusting Pho boxes response in both positive and negative manner under phosphorus limitation.     

Isolation of regulated genes of the cyanobacterium Synechocystis sp. strain PCC 6803 by differential display

Global identification of differentially regulated genes in prokaryotes is constrained because the mRNA does not have a 3' polyadenylation extension; this precludes specific separation of mRNA from rRNA and tRNA and synthesis of cDNAs from the entire mRNA population. Knowledge of the entire genome sequence of Synechocystis sp. strain PCC 6803 has enabled us to develop a differential display procedure that takes advantage of a short palindromic sequence that is dispersed throughout the Synechocystis sp. strain PCC 6803 genome. This sequence, designated the HIP (highly iterated palindrome) element, occurs in approximately half of the Synechocystis sp. strain PCC 6803 genes but is absent in rRNA and tRNA genes. To determine the feasibility of exploiting the HIP element, alone or in combination with specific primer subsets, for analyzing differential gene expression, we used HIP-based primers to identify light intensity-regulated genes. Several gene fragments, including those encoding ribosomal proteins and phycobiliprotein subunits, were differentially amplified from RNA templates derived from cells grown in low light or exposed to high light for 3 h. One novel finding was that expression of certain genes of the pho regulon, which are under the control of environmental phosphate levels, were markedly elevated in high light. High-light activation of pho regulon genes correlated with elevated growth rates that occur when the cells are transferred from low to high light. These results suggest that in high light, the rate of growth of Synechocystis sp. strain PCC 6803 exceeds its capacity to assimilate phosphate, which, in turn, may trigger a phosphate starvation response and activation of the pho regulon.     

集胞藻PCC6803铜离子诱导表达平台的构建

在集胞藻PCC6803中,基因敲除是研究基因功能的最直接有效的方法,但是对于某些生存必需的基因则无法通过这种方法获得突变株。为研究集胞藻PCC6803中此类基因的功能,在其基因组中构建了一个petE基因启动子（PpetE）控制的铜离子诱导表达的平台。将集胞藻PpetE装配在lacZ报告基因的上游,通过同源双交换整合到这种蓝藻的基因组中。通过调节培养基中铜离子的浓度发现,lacZ的表达能够人为控制。特别是当铜离子浓度在6－400nmoL／L范围时,LacZ活力随铜离子浓度增加呈S型增长关系。利用这个铜离子诱导表达平台,可以控制某些必需基因的表达：提供铜离子维持细胞生存;而撤去铜离子时则关闭基因的表达,可以观察其对生命活动的影响。     

General distribution of the nitrogen control gene ntcA in cyanobacteria.

The ntcA gene from Synechococcus sp. strain PCC 7942 encodes a regulatory protein which is required for the expression of all of the genes known to be subject to repression by ammonium in that cyanobacterium. Homologs to ntcA have now been cloned by hybridization from the cyanobacteria Synechocystis sp. strain PCC 6803 and Anabaena sp. strain PCC 7120. Sequence analysis has shown that these ntcA genes would encode polypeptides strongly similar (77 to 79% identity) to the Synechococcus NtcA protein. Sequences hybridizing to ntcA have been detected in the genomes of nine other cyanobacteria that were tested, including strains of the genera Anabaena, Calothrix, Fischerella, Nostoc, Pseudoanabaena, Synechococcus, and Synechocystis.     

Uptake of Pb2+ by a cyanobacterium belonging to the genus Synechocystis, isolated from East Kolkata Wetlands

East Kolkata Wetlands is a conserved wetland utilizing sewage and garbage, generated by Kolkata Municipal Corporation area for cultivation purpose. Cyanobacteria are the photosynthetic prokaryotes having bioremedial capacity. We have isolated a cyanobacterium from the sewage recycling fish-pond of East Kolkata Wetlands. Partial sequence of 16S rDNA gene of the isolated strain showed 100% similarity with that of genus Synechocystis. Isolated strain and Synechocystis sp. PCC6803 survived up to 300 mug ml(-1) Pb(2+ )and growth was completely inhibited at 400 mug ml(-1) Pb(2+). All experiments were carried out with 100 mug ml(-1) Pb(2+) in which growth was the maximum. 91.67% of the total Pb(2+) got adsorbed to the outer surface of the cell and 1% of the total Pb(2+) entered the cell of the isolated strain as estimated by atomic absorption spectrometry, but in Synechocystis sp. PCC6803 72.72% adsorbed and 0.96% penetrated. Intracellular and periplasmic depositions of Pb(2+) were observed in both the strain. A filamentous structure developed outside the cell wall of the isolated cyanobacterium, but very little change was observed in Synechocystis sp. PCC6803. ZiaR-SmtB like regulator gene was expressed in both the strains after Pb(2+) induction. The cDNA sequence of ZiaR of the isolated cyanobacterium shows 100% homology with that of Synechocystis sp. PCC6803. Upon Pb(2+) induction, expression of SOD gene increased. cDNA sequence of the SOD gene from the isolated strain showed 98% homology with that of Synechocystis sp. PCC6803. Enzymatic activity of catalase and SOD was also increased. No DNA damage was monitored upon induction with Pb(2+).     

Characterization of a two-component signal transduction system involved in the induction of alkaline phosphatase under phosphate-limiting conditions in Synechocystis sp. PCC 6803

The gene products of sll0337 and slr0081 in Synechocystis sp. PCC 6803 have been identified as the homologues of the Escherichia coli phosphate-sensing histidine kinase PhoR and response regulator PhoB, respectively. Interruption of sll0337, the gene encoding the histidine protein kinase, by a spectinomycin-resistance cassette blocked the induction of alkaline phosphatase activity under phosphate-limiting conditions. A similar result was obtained when slr0081, the gene encoding the response regulator, was interrupted with a cassette conferring resistance to kanamycin. In addition, the phosphate-specific transport system was not up-regulated in our mutants when phosphate was limiting. Unlike other genes for bacterial phosphate-sensing two-component systems, sll0337 and slr0081 are not present in the same operon. Although there are three assignments for putative alkaline phosphatase genes in the Synechocystis sp. PCC 6803 genome, only sll0654 expression was detected by northern analysis under phosphate limitation. This gene codes for a 149 kDa protein that is homologous to the cyanobacterial alkaline phosphatase reported in Synechococcus sp. PCC 7942 [Ray, J.M., Bhaya, D., Block, M.A. and Grossman, A.R. (1991) J. Bact. 173: 4297–4309]. An alignment identified a conserved 177 amino acid domain that was found at the N-terminus of the protein encoded by sll0654 but at the C-terminus of the protein in Synechococcus sp. PCC 7942.     

Circadian expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803.

The expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803 was continuously monitored as bioluminescence by an automated monitoring system, using the bacterial luciferase genes (luxAB) of Vibrio harveyi as a reporter of promoter activity. A dnaK-reporting bioluminescent Synechocystis strain was constructed by fusing a promoterless segment of the luxAB gene set downstream of the promoter region of the Synechocystis dnaK gene and introduction of this gene fusion into a BglII site downstream of the ndhB gene in the Synechocystis chromosome. Bioluminescence from this strain was continuously monitored and oscillated with a period of about 22 h for at least 5 days in continuous light. The phase of the rhythm was reset by the timing of the 12-h dark period administered prior to the continuous light. The period of the rhythm was temperature compensated between 25 and 35 degrees C. Thus, the bioluminescence rhythm satisfied the three criteria of circadian rhythms. Furthermore, the abundance of dnaK mRNA also oscillated with a period of about 1 day for at least 2 days in continuous light conditions, indicating circadian control of dnaK gene expression in Synechocystis sp. strain PCC 6803.