Active Fe-containing superoxide dismutase and abundant sodF mRNA in Nostoc commune (Cyanobacteria) after years of desiccation

Abstract

Active Fe-superoxide dismutase (SodF) was the third most abundant soluble protein in cells of Nostoc commune CHEN/1986 after prolonged (13 years) storage in the desiccated state. Upon rehydration, Fe-containing superoxide disumutase (Fe-SOD) was released and the activity was distributed between rehydrating cells and the extracellular fluid. The 21-kDa Fe-SOD polypeptide was purified, the N terminus was sequenced, and the data were used to isolate sodF from the clonal isolate N. commune DRH1. sodF encodes an open reading frame of 200 codons and is expressed as a monocistronic transcript (of approximately 750 bases) from a region of the genome which includes genes involved in nucleic acid synthesis and repair, including dipyrimidine photolyase (phr) and cytidylate monophosphate kinase (panC). sodF mRNA was abundant and stable in cells after long-term desiccation. Upon rehydration of desiccated cells, there was a turnover of sodF mRNA within 15 min and then a rise in the mRNA pool to control levels (quantity of sodF mRNA in cells in late logarithmic phase of growth) over approximately 24 h. The extensive extracellular polysaccharide (glycan) of N. commune DRH1 generated superoxide radicals upon exposure to UV-A or -B irradiation, and these were scavenged by SOD. Despite demonstrated roles for the glycan in the desiccation tolerance of N. commune, it may in fact be a significant source of damaging free radicals in vivo. It is proposed that the high levels of SodF in N. commune, and release of the enzyme from dried cells upon rehydration, counter the effects of oxidative stress imposed by multiple cycles of desiccation and rehydration during UV-A or -B irradiation in situ.

FIG. 1

(a) SodF is abundant in desiccated N. commune CHEN/1986. An immunoblot stained with Coomassie blue and used for N-terminal sequence analysis of SodF is shown. Wsp isoforms migrate between 30 and 39 kDa. The N-terminal sequence determined for the 21-kDa peptide (SodF) is indicated. (b) Distribution of sodF in the form species N. commune. An agarose gel (stained with ethidium bromide) of sodF PCR amplification products from samples of N. commune (Table 1) is shown. Lane M, HindIII molecular weight markers (LTI Inc.); lane 2, N. commune ALD8122/1974; lane 3, N. commune MEL/1968; lane 4, N. commune WH2/1939; lane 5, N. commune WH4/1869; lane 6, N. commune WH14/1948; lane 7, N. commune DRH1.

FIG. 2

A single Fe-SOD activity is identified in N. commune strains. Shown are SOD activities demonstrated in 7% (wt/vol) native tube gels with 0.7 U of N. commune ENG/1996 cell extract (lane 1), 1.3 U of N. commune ENG/1996 exudate (lane 2), 2.7 U of N. commune DRH1 cell extract (lane 3), and 2.4 U of N. commune DRH1 exudate (lane 4). The relative mobility (R_f value) was calculated by using the migration distance of the tracking dye as a reference.

FIG. 3

DNA sequence of sodF and flanking regions from N. commune DRH1. Shown is the complete coding region of sodF, with the putative amino acid sequence (underlined) and the putative ribosome-binding site (boldface underlining). Sequences of primers used to amplify the original 475-bp sodF fragment from genomic DNA correspond to positions 768 to 787 (5′ to 3′) and 1192 to 1210 (3′ to 5′) and are underlined; compare the N-terminal amino acid sequence with that of the native protein (Fig. 1a), which shows a single discrepancy (E→D) at position 16 of the open reading frame.

FIG. 4

SodF from N. commune DRH1 (SODF-NOSDRH1 [this study]) shows greatest sequence similarity to SodF from P. boryanum UTEX 485 (SODF-PLEBO [10]). The C-terminal regions of PanC proteins from Bacillus subtilis (KCY-BACSU), E. coli (KCY-ECOLI), and Haemophilus influenzae (KCY1-HAEIN) correspond to the partial sequence of PanC from N. commune DRH1 (KCY-NOSDRH1 [this study]). A partial alignment of Phr from Synechococcus sp. strain PCC 6301 (PHR-PCC6301) and N. commune DRH1 (PHR-NOSDRH1 [this study]) is shown. Sequences and alignments were manipulated with Lasergene software (DNASTAR, Madison, Wis.) and the software of the Biology Workbench, NCSA, University of Illinois (http://biology.ncsa.uiuc.edu/).

FIG. 5

(a) sodF expression in N. commune ENG/1996 by Northern blotting. Lane M contains RNA molecular weight markers (in kilobases). Other lanes contain mRNAs from colonies desiccated 3 years (lane 2) or rehydrated for 5 min (lane 3), 15 min (lane 4), 1 h (lane 5), 3 h (lane 6), 6 h (lane 7), 12 h (lane 8), or 24 h (lane 9). A liquid culture of N. commune DRH1 in late logarithmic phase of growth is shown in lane C. (b) 23S rRNA in N. commune ENG/1996 by Northern blotting. The lanes contain identical amounts of the same samples applied in panel a.

FIG. 6

Glycan of N. commune DRH1 generates O₂^−· when it is exposed to UV irradiation. (a) Glycan preparation A; (b) glycan preparation B. Shown is the reduction of ferricytochrome c (measured as increases in A₅₅₀) in the presence of glycan (▴), glycan plus SOD (○), glycan plus catalase (■), and the control (no glycan present in assay) (●). Values are determinations from single experiments; experiments were repeated four times, and the data presented are representative of those obtained in each trial. Variations in the A₅₅₀ at zero time are due to differences in the buffer solutions of the different components. The slopes of relevant curves were used to estimate the rates described in the text.