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Differential roles of alkaline/neutral invertases in Nostoc sp. PCC 7120: Inv-B isoform is essential for diazotrophic growth

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Abstract

The presence of two alkaline/neutral invertases (Inv-A and Inv-B) in the filaments of Nostoc (also named Anabaena) sp. strain PCC 7120 and the involvement of sucrose metabolism in nitrogen fixation led us to investigate the physiological function of those isoforms in cells growing under different nitrogen sources. The highest expression level of each encoding gene was obtained in the presence of ammonium. These results were paralleled by polypeptide and enzyme activity level. In cells of N2-fixing filaments, localization of gene expression and subcellular enzyme activity assays demonstrated that invA gene (alr1521) expresses only in vegetative cells, whereas for invB (alr0819), expression is detected in both vegetative cells and heterocysts. In contrast to invA, when invB was knocked out, the filaments were unable to grow on diazotrophic conditions and the accumulation of sucrose and glycogen was altered. Our results demonstrate an essential role for Inv-B for diazotrophic growth and that Inv-B plays a key role in the coordination of sucrose and glycogen metabolism. We can also suggest that invB is likely to integrate the repertoire of genes regulated by a cyanobacterial transcription factor (NtcA) that plays a central role in global nitrogen control.

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Abbreviations

A/N-Inv:

Alkaline/neutral invertase

Chl:

Chlorophyll

FW:

Fresh weight

Inv:

Invertase

gfp :

Green fluorescent protein encoding gene

Suc:

Sucrose

SPS:

Sucrose-phosphate synthase

SuS:

Sucrose synthase

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Acknowledgments

We are very thankful to L. Curatti, A. Cumino, and H.G. Pontis for helpful discussions, E. Etxeberria and P. Weyman for insightful reading of the manuscript, and C. Fernández for technical assistance. This research was funded by grants from CONICET, Universidad Nacional de Mar del Plata, ANPCyT (PICT No. 21227) and FIBA.

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  1. Walter A. Vargas

    Present address: Centro Hispano Luso de Investigaciones Agrarias, CIALE, Universidad de Salamanca, Salamanca, Spain

Authors and Affiliations

  1. Centro de Investigaciones Biológicas, CEBB-MdP-INBA, Fundación para Investigaciones Biológicas Aplicadas (FIBA), C.C. 1348, 7600, Mar del Plata, Argentina

    Walter A. Vargas, Carolina N. Nishi, Laura E. Giarrocco & Graciela L. Salerno

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  1. Walter A. Vargas
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  2. Carolina N. Nishi
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  3. Laura E. Giarrocco
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  4. Graciela L. Salerno
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Correspondence to Graciela L. Salerno.

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Supplemental Fig. 1

Generation of invA- and invB- mutant strains derivative from Nostoc sp. PCC 7120. a and b Genomic map depicting the construction of invA- and invB- insertion mutant strains, respectively. Cassettes coding for Em and Nm resistance were used to interrupt the coding region of invA or invB, respectively.c and d Electrophoresis of PCR-amplified DNA fragments confirming invA and invB interruption. Clones displaying resistance to Em or Nm were analyzed by PCR using the primer pair 366UEB/366LES or 182LES/182UEB, respectively. The former primer pair amplifies a 1,424-bp DNA fragment in the wild-type strain and a 3,424-bp DNA fragment in the invA- knockout strain. The latter primer pair amplifies a 1,469-bp DNA fragment in the wild-type strain and a fragment of 2,669 bp in the invB- knockout strain. e Schematic depicting of the construction of the invB+ mutant strain. It was generated cloning the coding region of invB in the plasmid pRL1404. The coding region of invB is transcribed from a promoter within the Nostoc replicon pDU1 (Walton et al., 1992). f Polypeptide levels determined in Nostoc sp. PCC 7120 (7120) and derivative mutant strains invA-, invB- and invB+. The three strains were grown in the presence of KNO3 as the source of combined nitrogen until the cultures reached an OD600 = 0.5. The samples were split for total protein extract preparations. A/N-Inv polypeptides were immunodetected by Western immunoblot analysis with anti-His6::Inv-B (Vargas et al. 2003). (TIFF 1432 kb)

Supplemental Fig. 2

Suc content (a and b) and Inv activity (c and d) in Nostoc sp. PCC 7120 (7120) and in derivative mutants (invA−, invB- and invB+). (TIFF 1138 kb)

Supplemental Fig. 3

Growth of Nostoc sp. PCC 7120 (7120) and derivative invA- mutant strain in different nitrogen sources. Cells were cultured in the presence of dinitrogen (a), NH4Cl (b) or KNO3 (c). Samples from each culture were collected every two days and optical density was determined. The plots represent the natural logarithm (ln) of the increase in optical densities vs days (based on the equation: ln (OD-OD0) = μ.t + ln e). Similar plots were obtained when growth curves were based on fresh weight or chlorophyll content. (TIFF 1163 kb)

Supplemental Fig. 4

Morphology of the filaments of the Nostoc sp. PCC 7120 (7120), invA-, invB- and invB+ strains grown in diazotrophic conditions or in the presence of NH4Cl for 10 days. Bright field images were photographed and processed with same settings and were taken at the same magnification (1,000x), to allow qualitative comparison of the filaments. Arrowheads indicate heterocysts. (TIFF 2671 kb)

Supplemental Fig. 5

Position of genes related to Suc metabolism in the genome of Nostoc sp. PCC 7120. Genes encoding proteins involved in Suc synthesis (spsA and spsB) and degradation (susA, invA, and invB). The imaginary line dividing the genome (265º - 85º) in two halves suggested by Sugaya et al. (2004) is indicated with a green dotted line. (TIFF 7502 kb)

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Vargas, W.A., Nishi, C.N., Giarrocco, L.E. et al. Differential roles of alkaline/neutral invertases in Nostoc sp. PCC 7120: Inv-B isoform is essential for diazotrophic growth. Planta 233, 153–162 (2011). https://doi.org/10.1007/s00425-010-1288-5

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