Molecular and genetic analysis of two closely linked genes that encode, respectively, a protein phosphatase 1/2A/2B homolog and a protein kinase homolog in the cyanobacterium Anabaena sp. strain PCC 7120 - PubMed (original) (raw)

Molecular and genetic analysis of two closely linked genes that encode, respectively, a protein phosphatase 1/2A/2B homolog and a protein kinase homolog in the cyanobacterium Anabaena sp. strain PCC 7120

C C Zhang et al. J Bacteriol. 1998 May.

Abstract

Reversible protein phosphorylation plays important roles in signal transduction. One gene, prpA, encoding a protein similar to eukaryotic types of phosphoprotein phosphatases PP1, PP2A, and PP2B, was cloned from the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. Interestingly, a eukaryotic-type protein kinase gene, pknE, was found 301 bp downstream of prpA. This unusual genetic arrangement provides the opportunity for study about how the balance between protein phosphorylation and dephosphorylation can regulate cellular activities. Both proteins were overproduced in Escherichia coli and used to raise polyclonal antibodies. Immunodetection and RNA/DNA hybridization experiments suggest that these two genes are unlikely to be coexpressed, despite their close genetic linkage. PrpA is expressed constitutively under different nitrogen conditions, while PknE expression varies according to the nature of the nitrogen source. Inactivation analysis in vivo suggests that PrpA and PknE function to ensure a correct level of phosphorylation of the targets in order to regulate similar biological processes such as heterocyst structure formation and nitrogen fixation.

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Figures

FIG. 1

FIG. 1

Restriction map of the prpA-pknE region. The coding regions of the two genes are shown by arrow bars below the restriction map. Shaded regions correspond to the catalytic domains of PrpA and PknE. DNA fragments (pHE10, pPPE3, pp-1, and pk-1) relevant to the text are also shown. The strategies used to inactivate either the prpA gene or the pknE gene are illustrated above the restriction map (for details, see Materials and Methods).

FIG. 2

FIG. 2

Amino acid sequence comparison between PrpA and Ser/Thr phosphatases (A) and PknE and Ser/Thr kinases (B). Identical residues are in bold-faced type. Gaps (indicated by dashes) were introduced to optimize sequence alignment. PP1-AT, protein phosphatase 1 in A. thaliana (23); PP2A-hu, human protein phosphatase 2A (25); PP2B-DM, protein phosphatase 2B in D. melanogaster (15); PknA, protein kinase in Anabaena (29); Raf-rat, the Ser/Thr kinase Raf in rats (18).

FIG. 3

FIG. 3

Hydrolysis of pNPP by the recombinant GST-PrpA fusion protein. The affinity-column-purified proteins (GST and GST-PrpA fusion) were incubated at 30°C in the presence of different ions. The hydrolysis of pNPP was followed by measurement of the change in OD410 (22). ⊡, GST-PrpA with 2 mM Mn2+; formula image, GST with 2 mM Mn2+; formula image, GST-PrpA with 2 mM Ca2+; and ▵, GST-PrpA with 2 mM Mg2+.

FIG. 4

FIG. 4

Expression of PrpA and PknE examined by Western blotting. Total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto Immobilon membrane for Western analysis with antibodies against either PrpA (A and C) or PknE (B and D). To check the protein expression during heterocyst development (A and B), similar amounts of total proteins were loaded onto each lane of the gel. Protein samples were prepared from cells grown in the presence of nitrate (lanes 0, NO3) or transferred to nitrate-free medium (N2) for 1 to 96 h (lanes 1 to lanes 96).

FIG. 5

FIG. 5

Growth curves of the wild type (▪), the prpA mutant A.3 (□), and the pknE mutant 8.1 (⧫) under N2-fixing conditions. Cells were incubated in a combined nitrogen-free medium (BG110) at 30°C in air. Cell growth was monitored by measuring the OD700.

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