Effect of nitrate availability on expression of multi sensor histidine kinase gene in Spirulina platensis PCC 7345

Abstract

Addition of nitrate (NO3 (-)) to a suspension of NO3 (-)-depleted Spirulina platensis PCC 7345 restored back the PSII reaction centre efficiency. Using RAPD/RT-PCR differential display, we have detected the upregulation of a novel gene in filaments starved of nitrogen when enriched with NO3 (-). Semiquantitative RT-PCR clearly shows that NO3 (-) repletion induces the expression of this gene. Its nucleotide sequence is homologous to multi sensor histidine kinase (hstk) from Spirulina platensis NIES-39 and Spirulina maxima CS-328 genes. The open reading frame of this gene encodes a protein of 98 amino acids contains sensory domain of PAS domain.

Keywords: Differential display; Histidine kinase; PSII reaction centre efficiency; Spirulina.

Fig. 1

Fluorescence emission spectra of a) cells and b) isolated thylakoid membrane. Cells grown in nitrate free medium were used as control against the cells grown in 10 mM KNO₃ for 2 hrs. The fluorescence spectra were recorded on equal Chl a basis (5 μg ml⁻¹) at excitation wavelength of 440 nm. These experiments were done in triplicates yielded identical spectra

Fig. 2

a RAPD RT-PCR with primer sets giving promising cDNA pattern visualized by EtBr staining; b Differential display gel showing cDNAs expressed only in NO₃⁻ repleted filaments: [NO₃⁻ depleted controls marked with (−) and NO₃⁻ repleted filaments marked with (+), superscript to the primer combinations used]. c Differentially expressed cDNA was eluted from the gel and reamplified using primers DD2 and DD5 for sequencing

Fig. 3

Semi-quantitative RT-PCR analysis of hstk (283 bp) and 16 S rRNA (479 bp). [Lane 1: marker; lane 2: no RNA control; lane 3: 16S rRNA from nitrate treated cells; lane 4: 16S rRNA from non-treated; lane 5: hstk from nitrate treated cells; lane 6: hstk from non-treated]. The bar graph shows the amount of mRNA, expressed as the ratio of densitometric measurement. Data are shown as mean±SEM for 3 independent experiments, *p < 0.05 vs lane 6 (NO₃⁻ Depleted)

Fig. 4

a Sequence alignment between HstK of S. maxima CS-328, S. platensis PCC 7345 and S. platensis NIES-39. b The putative domain of the protein encoded by HstK