Skip to main content
SpringerLink
Log in

Alteration of the exopolysaccharide production and the transcriptional profile of free-living Frankia strain CcI3 under nitrogen-fixing conditions

  • Applied microbial and cell physiology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

We investigated the effect of different nitrogen (N) sources on exopolysaccharide (EPS) production and composition by Frankia strain CcI3, a N2-fixing actinomycete that forms root nodules with Casuarina species. Frankia cells grown in the absence of NH4Cl (i.e., under N2-fixing conditions) produced 1.7-fold more EPS, with lower galactose (45.1 vs. 54.7 mol%) and higher mannose (17.3 vs. 9.7 mol%) contents than those grown in the presence of NH4Cl as a combined N-source. In the absence of the combined N-source, terminally linked and branched residue contents were nearly twice as high with 32.8 vs. 15.1 mol% and 15.1 vs. 8.7 mol%, respectively, than in its presence, while the content of linearly linked residues was lower with 52.1 mol% compared to 76.2 mol%. To find out clues for the altered EPS production at the transcriptional level, we performed whole-gene expression profiling using quantitative reverse transcription PCR and microarray technology. The transcription profiles of Frankia strain CcI3 grown in the absence of NH4Cl revealed up to 2 orders of magnitude higher transcription of nitrogen fixation-related genes compared to those of CcI3 cells grown in the presence of NH4Cl. Unexpectedly, microarray data did not provide evidence for transcriptional regulation as a mechanism for differences in EPS production. These findings indicate effects of nitrogen fixation on the production and composition of EPS in Frankia strain CcI3 and suggest posttranscriptional regulation of enhanced EPS production in the absence of the combined N-source.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Agarwal AK, Keister L (1983) Physiology of ex-planta nitrogenase activity in Rhizobium japonicum. Appl Environ Microbiol 45:1592–1601

    PubMed  CAS  Google Scholar 

  • Alloisio N, Felix S, Marechal J, Pujic P, Rouy Z, Vallenet D, Medigue C, Normand P (2007) Frankia alni proteome under nitrogen-fixing and nitrogen-replete conditions. Physiol Plant 130:440–453

    Article  CAS  Google Scholar 

  • Alloisio N, Queiroux C, Fournier P, Pujic P, Normand P, Vallenet D, Médigue C, Yamaura M, Kakoi K, Kucho K (2010) The Frankia alni symbiotic transcriptome. Mol Plant-Microbe Interact 23:593–607

  • Amann RI, Krumholz L, Stahl DA (1990a) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770

    PubMed  CAS  Google Scholar 

  • Amann RI, Binder BJ, Olsen RJ, Chisholm SW, Devereux R, Stahl DA (1990b) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925

    PubMed  CAS  Google Scholar 

  • Arnone JA III, Gordon JC (1997) Evidence for N feedback regulation of N2 fixation in Alnus glutinosa L. J Exp Bot 48:67–73

    Article  Google Scholar 

  • Arnone JAI, Kohls SJ, Baker DD (1994) Nitrate effects on nodulation and nitrogenase activity of actinorhizal Casuarina studied in split-root systems. Soil Biol Biochem 26:599–606

    Google Scholar 

  • Baker D, O'Keefe D (1984) A modified sucrose fractionation procedure for the isolation of frankiae from actinorhizal root-nodules and soil samples. Plant Soil 78:23–28

    Article  Google Scholar 

  • Benson DR, Silvester WB (1993) Biology of Frankia strains, actinomycete symbionts of actinorhizal plants. Microbiol Rev 57:293–319

    PubMed  CAS  Google Scholar 

  • Beverley SM, Owens KL, Showalter M, Griffith CL, Doering TL, Jones VC, McNeil MR (2005) Eukaryotic UDP-galactopyranose mutase (GLF gene) in microbial and metazoal pathogens. Eukaryot Cell 4:1147–1154

    Google Scholar 

  • Bhagwat AA, Mithofer A, Pfeffer PE, Kraus C, Spickers N, Hotchkiss A, Ebel J, Keister DL (1999) Further studies of the role of cyclic β-glucans in symbiosis. An ndvC mutant of Bradyrhizobium japonicum synthesizes cyclodecakis-(1 → 3)-β-glucosyl. Plant Physiol 119:1057–1064

    Article  PubMed  CAS  Google Scholar 

  • Bickhart DM, Benson DR (2011) Transcriptomes of Frankia sp. strain CcI3 in growth transitions. BMC Microbiol 11:192–208

    Article  PubMed  CAS  Google Scholar 

  • Bond G, Mackintosh AH (1975) Effect of nitrate-nitrogen on the nodule symbioses of Coriaria and Hippophae. P Roy Soc Lond B Bio 190:199–209

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Brelles-Marino G, Boiardi JL (1996) Nitrogen limitation of chemostat-grown Rhizobium etli elicits higher infection-thread formation in Phaseolus vulgaris. Microbiology 142:1067–1070

    Article  CAS  Google Scholar 

  • Callaham D, Deltredici P, Torrey JG (1978) Isolation and cultivation in vitro of actinomycete causing root nodulation in Comptonia. Science 199:899–902

    Article  PubMed  CAS  Google Scholar 

  • Chang W-S, van de Mortel M, Nielsen L, de Guzman GN, Li X, Halverson LJ (2007) Alginate production by Pseudomonas putida creates a hydrated microenvironment and contributes to biofilm architecture and stress tolerance under water-limiting conditions. J Bacteriol 189:8290–8299

    Google Scholar 

  • Crannell WK, Tanaka Y, Myrold DD (1994) Calcium and pH interaction on root nodulation of nursery-grown red alder (Alnus rubra Bong.) seedlings by Frankia. Soil Biol Biochem 26:607–614

    Article  CAS  Google Scholar 

  • Cytryn EJ, Sangurdekar DP, Streeter JG, Franck WL, Chang W-S, Stacey G, Emerich DW, Joshi T, Xu D, Sadowsky MJ (2007) Transcriptional and physiological responses of Bradyrhizobium japonicum to desiccation-induced stress. J Bacteriol 189:6751–6762

    Article  PubMed  CAS  Google Scholar 

  • Dawson JO, Kowalski DG, Dart PJ (1989) Variation with soil depth, topographic position and host species in the capacity of soils from an Australian locale to nodulate Casuarina and Allocasuarina seedlings. Plant Soil 118:1–11

    Article  Google Scholar 

  • Donati AJ, Jeon J-M, Sangurdekar D, So J-S, Chang W-S (2011) Genome-wide transcriptional and physiological responses of Bradyrhizobium japonicum to paraquat-mediated oxidative stress. Appl Environ Micobiol 77:3633–3643

    Article  CAS  Google Scholar 

  • Dubois M, Giles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356

    Article  CAS  Google Scholar 

  • Dunlap J, Minami E, Bhagwat AA, Keister DL, Stacey G (1996) Nodule development induced by mutants of Bradyrhizobium japonicum defective in cyclic β-glucan synthesis. Mol Plant-Microbe Interact 9:546–555

    Google Scholar 

  • Gauthier D, Diem HG, Dommergues Y (1981) In vitro nitrogen fixation by two actinomycete strains isolated from Casuarina nodules. Appl Environ Microbiol 41:306–308

    PubMed  CAS  Google Scholar 

  • Gentili F, Huss-Danell K (2003) Local and systemic effects of phosphorus and nitrogen on nodulation and nodule function in Alnus incana. J Exp Bot 54:2757–2767

    Article  PubMed  CAS  Google Scholar 

  • Gonzalez JE, Reuhs BL, Walker GC (1996) Low molecular weight EPS II of Rhizobium meliloti allows nodule invasion in Medicago sativa. Proc Natl Acad Sci USA 93:8636–8641

    Article  PubMed  CAS  Google Scholar 

  • Griffiths AP, McCormick LH (1984) Effects of soil acidity on nodulation of Alnus glutinosa and viability of Frankia. Plant Soil 79:429–434

    Article  CAS  Google Scholar 

  • Hahn D, Zepp K, Zeyer J (1997) Whole cell hybridization as a tool to study Frankia populations in root nodules. Plant Physiol 99:696–706

    Article  CAS  Google Scholar 

  • Hellsten A, Huss-Danell K (2001) Interaction effects of nitrogen and phosphorus on nodulation in red clover (Trifolium pratense L.). Acta Agr Scand B-S P 50:135–142

    Google Scholar 

  • Hotter GS, Scott DB (1991) Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host but are ineffective on an indeterminate nodulating host. J Bacteriol 173:851–859

    PubMed  CAS  Google Scholar 

  • Huss-Danell K (1997) Actinorhizal symbioses and their N2 fixation. New Phytol 136:375–405

    Article  CAS  Google Scholar 

  • Jain DK, Prevost D, Bordeleau LM (1990) Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia. FEMS Microbiol Ecol 73:167–173

    Article  CAS  Google Scholar 

  • Kohls SJ, Baker DD (1989) Effects of substrate nitrate concentration on symbiotic nodule formation in actinorhizal plants. Plant Soil 118:171–179

    Article  CAS  Google Scholar 

  • Koplin R, Brisson JR, Whitfield C (1997) UDP-galactofuranose precursor required for formation of the lipopolysaccharide O antigen of Klebsiella pneumoniae serotype O1 is synthesized by the product of the rfbDKPO 1 gene. J Biol Chem 272:4121–4128

    Article  PubMed  CAS  Google Scholar 

  • Kucho K, Hay AE, Normand P (2010) The determinants of the actinorhizal symbiosis. Microbes Environ 25:241–252

    Article  PubMed  Google Scholar 

  • Lopez-Garcia SL, Vazquez TE, Favelukes G, Lodeiro AR (2001) Improved soybean root association of N-starved Bradyrhizobium japonicum. J Bacteriol 183:7241–7252

    Article  PubMed  CAS  Google Scholar 

  • Mastronunzio JE, Benson DR (2010) Wild nodules can be broken: proteomics of Frankia in field-collected root nodules. Symbiosis 50:13–26

    Article  CAS  Google Scholar 

  • Mastronunzio JE, Huang Y, Benson DR (2009) Diminished exoproteome of Frankia spp. in culture and symbiosis. Appl Environ Microbiol 75:6721–6728

    Article  PubMed  CAS  Google Scholar 

  • Meesters TM, Vangenesen ST, Akkermans ADL (1985) Growth, acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc117 and Cp12. Arch Microbiol 143:137–142

    Article  CAS  Google Scholar 

  • Merkle RK, Poppe I (1994) Carbohydrate composition analysis of glycoconjugates by gas–liquid chromatography/mass spectrometry. Meth Enzymol 230:1–15

    Article  PubMed  CAS  Google Scholar 

  • Normand P, Lapierre P, Tisa LS, Gogarten JP, Alloisio N, Bagnarol E, Bassi CA, Berry AM, Bickhart DM, Choisne N, Couloux A, Cournoyer B, Cruveiller S, Daubin V, Demange N, Francino MP, Goltsman E, Huang Y, Kopp OR, Labarre L, Lapidus A, Lavire C, Marechal J, Martinez M, Mastronunzio JE, Mullin BC, Niemann J, Pujic P, Rawnsley T, Rouy Z, Schenowitz C, Sellstedt A, Tavares F, Tomkins J, Vallenet D, Valverde C, Wall LG, Wang Y, Medigue C, Benson DR (2007) Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography. Genome Res 17:7–15

    Article  PubMed  Google Scholar 

  • Parniske M, Kosch K, Werner D, Müller P (1993) exoB mutants of Bradyrhizobium japonicum with reduced competitiveness for nodulation of Glycine max. Mol Plant Microbe Interact 6:99–106

    Article  CAS  Google Scholar 

  • Parsons R, Silvester WB, Harris S, Gruijters WTM, Bullivant S (1987) Frankia vesicles provide inducible and absolute oxygen protection for nitrogenase. Plant Physiol 83:728–731

    Article  PubMed  CAS  Google Scholar 

  • Peltier P, Euzen R, Daniellou R, Nugier-Chauvin C, Ferrieres V (2008) Recent knowledge and innovations related to hexofuranosides: structure, synthesis and applications. Carbohydr Res 343:1897–1923

    Article  PubMed  CAS  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007

    Article  Google Scholar 

  • Quelas JI, López-García SL, Casabuono A, Althabegoiti MJ, Mongiardini EJ, Pérez-Giménez J, Couto A, Lodeiro AR (2006) Effects of N-starvation and C-source on Bradyrhizobium japonicum exopolysaccharide production and composition, and bacterial infectivity to soybean roots. Arch Microbiol 186:119–128

    Article  PubMed  CAS  Google Scholar 

  • Ramakers C, Ruijter JM, Deprez RH, Moorman AF (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66

    Article  PubMed  CAS  Google Scholar 

  • Read RR, Costerton JW (1987) Purification and characterization of adhesive exopolysaccharides from Pseudomonas putida and Pseudomonas fluorescens. Can J Microbiol 33:1080–1090

    Article  PubMed  CAS  Google Scholar 

  • Richards MR, Lowary TL (2009) Chemistry and biology of galactofuranose-containing polysaccharides. Chembiochem 10:1920–1938

    Article  PubMed  CAS  Google Scholar 

  • Sanders DAR, Staines AG, McMahon SA, McNeil MR, Whitfield C, Naismith JH (2001) UDP-galactopyranose mutase has a novel structure and mechanism. Nat Struct Biol 8:858–863

    Article  PubMed  CAS  Google Scholar 

  • Sanginga N, Danso SKA, Bowen GD (1989) Nodulation and growth response of Allocasuarina and Casuarina species to phosphorus fertilization. Plant Soil 118:125–132

    Article  Google Scholar 

  • Schultz NA, Benson DR (1990) Enzymes of ammonia assimilation in hyphae and vesicles of Frankia sp. Strain CpI1. J Bacteriol 172:1380–1384

    PubMed  CAS  Google Scholar 

  • Schwintzer CR (1985) Effect of spring flooding on endophyte differentiation, nitrogenase activity, root growth and shoot growth in Myrica gale. Plant Soil 87:109–124

    Article  CAS  Google Scholar 

  • Schwintzer CR (1990) Spore-positive and spore-negative nodules. In: Tjepkema JD, Schwintzer CR (eds) The biology of Frankia and actinorhizal plants. Academic, San Diego, pp 178–195

    Google Scholar 

  • Stewart WDP, Bond G (1961) The effect of ammonium nitrogen on fixation of elemental nitrogen in Alnus and Myrica. Plant Soil 14:347–359

    Article  CAS  Google Scholar 

  • St-Laurent L, Bousquet J, Simon L, Lalonde M (1987) Separation of various Frankia strains in the Alnus and Elaeagnus host specificity groups using sugar analysis. Can J Microbiol 33:764–772

    Article  CAS  Google Scholar 

  • Taniuchi Y, Murakami A, Ohki K (2008) Whole-cell immunocytochemical detection of nitrogenase in cyanobacteria: improved protocol for highly fluorescent cells. Aquat Microb Ecol 51:237–247

    Article  Google Scholar 

  • Thomas KA, Berry AM (1989) Effects of continuous nitrogen application and nitrogen preconditioning on nodulation and growth of Ceanothus griseus var. horizontalis. Plant Soil 118:181–187

    Article  Google Scholar 

  • Tjepkema JD, Ormerod W, Torrey JG (1980) Vesicle formation and acetylene reduction activity in Frankia sp. Cpi1 cultured in defined nutrient media. Nature 287:633–635

    Article  CAS  Google Scholar 

  • Weston A, Sternt RJ, Lee RE, Nassau PM, Monsey D, Martin SL, Scherman MS, Besra GS, Duncan K, McNeil MR (1998) Biosynthetic origin of mycobacterial cell wall galactofuranosyl residues. Tuber Lung Dis 78:123–131

    Article  Google Scholar 

  • Yang Y (1995) The effect of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis. Plant Soil 176:161–169

    Article  CAS  Google Scholar 

  • York WS, Darvill AG, McNeil M, Stevenson TT, Albersheim P (1985) Isolation and characterization of plant cell walls and cell wall components. Meth Enzymol 118:3–40

    Article  Google Scholar 

  • Zhang XJ, Benson DR (1992) Utilization of amino acids by Frankia sp. strain Cpi1. Arch Microbiol 158:256–261

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Raymond Jones in the Genomics Core Facility at the University at Texas-Arlington for providing the technical support for the qRT-PCR analysis and Dr. Schwartz, senior scientist from Mycroarray.com, for his assistance with the microarray data analysis. This research was supported by a Research Enhancement Program (REP) grant from the University of Texas-Arlington (WSC) and in part by Hatch grant NH530 (LST).

Author information

Authors and Affiliations

  1. Department of Biology, University of Texas, Arlington, TX, 76019, USA

    Hae-In Lee, Andrew J. Donati & Woo-Suk Chang

  2. Department of Biology, Texas State University, San Marcos, TX, 78666, USA

    Dittmar Hahn

  3. Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, USA

    Louis S. Tisa

Authors
  1. Hae-In Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew J. Donati
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dittmar Hahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Louis S. Tisa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Woo-Suk Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Woo-Suk Chang.

Additional information

Hae-In Lee and Andrew J. Donati contributed equally.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 194 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, HI., Donati, A.J., Hahn, D. et al. Alteration of the exopolysaccharide production and the transcriptional profile of free-living Frankia strain CcI3 under nitrogen-fixing conditions. Appl Microbiol Biotechnol 97, 10499–10509 (2013). https://doi.org/10.1007/s00253-013-5277-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-013-5277-z

Keywords

Search

Navigation