Skip to main content
SpringerLink
Log in

Isolation and characterization of active promoters from Gluconacetobacter diazotrophicus strain PAL5 using a promoter-trapping plasmid

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

Gluconacetobacter diazotrophicus is a nitrogen-fixing, endophytic bacterium that has the potential to promote plant growth and increase yield. Genetically modified strains might get more benefits to host plants, including through expression of useful proteins, such as Cry toxins from B. thuringiensis, or enzymes involved in phytohormone production, proteins with antagonistic activity for phytopathogens, or that improve nutrient utilization by the plant. For that, expression systems for G. diazotrophicus are needed, which requires active promoters fused to foreign (or innate) genes. This article describes the construction of a G. diazotrophicus PAL5 promoter library using a promoter-less lacZ-bearing vector, and the identification of six active promoters through β-galactosidase activity assays, sequencing and localization in the bacterial genome. The characterized promoters, which are located on distinct regions of the bacterial genome and encoding either sense or antisense transcripts, present variable expression strengths and might be used in the future for expressing useful proteins.

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
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Alquéres SM, Cardoso AM, Brito-Moreira J et al (2012) Transfer RNA-dependent asparagine biosynthesis in Gluconacetobacter diazotrophicus and its influence on biological nitrogen fixation. Plant Soil 356:209–216

    Article  Google Scholar 

  • Alquéres S, Meneses CHSG, Rouws L et al (2013) The bacterial superoxide dismutase and gluthatione reductase are crucial for endophytic colonization of rice roots by Gluconacetobacter diazotrophicus strain PAL5. Mol Plant Microbe Interact 26:937–945

    Article  PubMed  Google Scholar 

  • Baldani JI, Baldani VLD (2005) History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience. An Acad Bras Ciênc 77:549–579

    Article  CAS  PubMed  Google Scholar 

  • Baldani J, Krieg NR, Baldani VLD et al (2005) Genus II. Azospirillum. In: Brenner DJ, Krieg NR, Staley JT (eds) Bergey’s manual® of systematic bacteriology, 2nd edn. Springer, New York, pp 22–24

  • Barbosa EA, Perin L, Reis VM (2006) Uso de diferentes fontes de carbono por estirpes de Gluconacetobacter diazotrophicus isoladas de cana-de-açúcar. Pesqui Agropecu Bras 41:827–833

    Article  Google Scholar 

  • Bastián F, Cohen A, Piccoli P et al (1998) Production of indole-3-acetic acid and gibberellins A1 and A3 by Acetobacter diazotrophicus and Herbaspirillum seropedicae in chemically-defined culture media. Plant Growth Regul 24:7–11

    Article  Google Scholar 

  • Bertalan M, Albano R, de Pádua V et al (2009) Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5. BMC Genom 10:450

    Article  Google Scholar 

  • Bertini EV, Peñalver CGN, Leguina AC et al (2014) Gluconacetobacter diazotrophicus PAL5 possesses an active quorum sensing regulatory system. Antonie Van Leeuwenhoek 106:497–506

    Article  CAS  PubMed  Google Scholar 

  • Blanco Y, Blanch M, Piñón D et al (2005) Antagonism of Gluconacetobacter diazotrophicus (a sugarcane endosymbiont) against Xanthomonas albilineans (pathogen) studied in alginate-immobilized sugarcane stalk tissues. J Biosci Bioeng 99:366–371

    Article  CAS  PubMed  Google Scholar 

  • Boddey RM, Urquiaga S, Alves BJR, Reis V (2003) Endophytic nitrogen fixation in sugarcane: present knowledge and future applications. Plant Soil 252:139–149

    Article  CAS  Google Scholar 

  • Cavalcante VA, Döbereiner J (1988) A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane. Plant Soil 108:23–31

    Article  Google Scholar 

  • Clarke L, Carbon J (1976) A colony bank containing synthetic CoI EI hybrid plasmids representative of the entire E. coli genome. Cell 9:91–99

    Article  CAS  PubMed  Google Scholar 

  • de Paula Soares C, Rodrigues EP, de Paula Ferreira J et al (2015) Tn5 insertion in the tonB gene promoter affects iron-related phenotypes and increases extracellular siderophore levels in Gluconacetobacter diazotrophicus. Arch Microbiol 197:223–233

    Article  Google Scholar 

  • Dong Z, Heydrich M, Bernard K, McCully ME (1995) Further evidence that the N2-fixing endophytic bacterium from the intercellular spaces of sugarcane stems is Acetobacter diazotrophicus. Appl Environ Microbiol 61:1843–1846

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dornenburg JE, DeVita AM, Palumbo MJ, Wade JT (2010) Widespread antisense transcription in Escherichia coli. MBio 1:e00024-10

    Article  PubMed  PubMed Central  Google Scholar 

  • Doyle JJ (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  • Georg J, Hess WR (2011) cis-antisense RNA, another level of gene regulation in bacteria. Microbiol Mol Biol Rev 75:286–300

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gillis M, Kersters K, Hoste B et al (1989) Acetobacter diazotrophicus sp. nov., a nitrogen-fixing acetic acid bacterium associated with sugarcane. Int J Syst Bacteriol 39:361–364

    Article  Google Scholar 

  • Giongo A, Tyler HL, Zipperer UN, Triplett EW (2010) Two genome sequences of the same bacterial strain, Gluconacetobacter diazotrophicus PAl 5, suggest a new standard in genome sequence submission. Stand Genomic Sci 2:309–317. doi:10.4056/sigs.972221

    Article  PubMed  PubMed Central  Google Scholar 

  • Griffith KL, Wolf RE (2002) Measuring β-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays. Biochem Biophys Res Commun 290:397–402

    Article  CAS  PubMed  Google Scholar 

  • Haapalainen M, Karp M, Metzler MC (1996) Isolation of strong promoters from Clavibacter xyli subsp. cynodontis using a promoter probe plasmid. Biochim Biophys Acta 1305:130–134

    Article  PubMed  Google Scholar 

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    Article  CAS  PubMed  Google Scholar 

  • Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2898–2989

    Article  Google Scholar 

  • Kallnik V, Meyer M, Deppenmeier U, Schweiger P (2010) Construction of expression vectors for protein production in Gluconobacter oxydans. J Biotechnol 150:460–465

    Article  CAS  PubMed  Google Scholar 

  • Li RP, MacRae IC (1991) Specific association of diazotrophic acetobacters with sugarcane. Soil Biol Biochem 23:999–1002

    Article  CAS  Google Scholar 

  • Lisser S, Margalit H (1993) Compilation of E. coli mRNA promoter sequences. Nucleic Acids Res 21:1507–1516

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Logeshwaran P, Thangaraju M, Rajasundari K (2009) Hydroxamate siderophores of endophytic bacteria Gluconacetobacter diazotrophicus isolated from sugarcane roots. Aust J Basic Appl Sci 3:3564–3567

    CAS  Google Scholar 

  • Maheshkumar KS, Krishnaraj PU, Alagawadi AR (1999) Mineral phosphate solubilizing activity of Acetobacter diazotrophicus: a bacterium associated with sugarcane. Curr Sci 76:874–875

    Google Scholar 

  • Meneses CHSG, Rouws LFM, Simões-Araújo JL et al (2011) Exopolysaccharide production is required for biofilm formation and plant colonization by the nitrogen-fixing endophyte Gluconacetobacter diazotrophicus. Mol Plant Microbe Interact 24:1448–1458

    Article  CAS  PubMed  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Oliveira ALM, Urquiaga S, Döbereiner J, Baldani JI (2002) The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants. Plant Soil 242:205–215. doi:10.1023/A:1016249704336

    Article  CAS  Google Scholar 

  • Oliveira ALM, Canuto EL, Urquiaga S et al (2006) Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria. Plant Soil 284:23–32. doi:10.1007/s11104-006-0025-0

    Article  CAS  Google Scholar 

  • Pothier JF, Wisniewski-Dyé F, Weiss-Gayet M et al (2007) Promoter-trap identification of wheat seed extract-induced genes in the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp245. Microbiology 153:3608–3622

    Article  CAS  PubMed  Google Scholar 

  • Ramírez-Romero MA, Masulis I, Cevallos MA et al (2006) The Rhizobium etli σ70 (SigA) factor recognizes a lax consensus promoter. Nucleic Acids Res 34:1470–1480

    Article  PubMed  PubMed Central  Google Scholar 

  • Rapulana T, Bouwer G (2013) Toxicity to Eldana saccharina of a recombinant Gluconacetobacter diazotrophicus strain carrying a truncated Bacillus thuringiensis cry1Ac gene. Afr J Microbiol Res 7:1207–1214

    CAS  Google Scholar 

  • Rodrigues Neto J, Malavolta VA Jr, Victor O (1986) Meio simples para o isolamento e cultivo de Xanthomonas campestris pv. citri tipo B. Summa Phytopathol 12:32

    Google Scholar 

  • Rouws LFM, Simões-Araújo JL, Hemerly AS, Baldani JI (2008) Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant. Arch Microbiol 189:397–405

    Article  CAS  PubMed  Google Scholar 

  • Rouws LFM, Meneses CHSG, Guedes HV et al (2010) Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes. Lett Appl Microbiol 51:325–330. doi:10.1111/j.1472-765X.2010.02899.x

    Article  CAS  PubMed  Google Scholar 

  • Rowland B, Purkayastha A, Monserrat C et al (1999) Fluorescence-based detection of lacZ reporter gene expression in intact and viable bacteria including Mycobacterium species. FEMS Microbiol Lett 179:317–325

    Article  CAS  PubMed  Google Scholar 

  • Saichana N, Matsushita K, Adachi O et al (2014) Acetic acid bacteria: a group of bacteria with versatile biotechnological applications. Biotechnol Adv. doi:10.1016/j.biotechadv.2014.12.001

    PubMed  Google Scholar 

  • Saravanan VS, Kalaiarasan P, Madhaiyan M, Thangaraju M (2007) Solubilization of insoluble zinc compounds by Gluconacetobacter diazotrophicus and the detrimental action of zinc ion (Zn2+) and zinc chelates on root knot nematode Meloidogyne incognita. Lett Appl Microbiol 44:235–241

    Article  CAS  PubMed  Google Scholar 

  • Schlüter J-P, Reinkensmeier J, Daschkey S et al (2010) A genome-wide survey of sRNAs in the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti. BMC Genom 11:245

    Article  Google Scholar 

  • Schwab S, Ramos HJ, Souza EM et al (2007) Identification of NH4 +-regulated genes of Herbaspirillum seropedicae by random insertional mutagenesis. Arch Microbiol 187:379–386

    Article  CAS  PubMed  Google Scholar 

  • Sesto N, Wurtzel O, Archambaud C et al (2012) The excludon: a new concept in bacterial antisense RNA-mediated gene regulation. Nat Rev Microbiol 11:75–82

    Article  PubMed  Google Scholar 

  • Sevilla M, Burris RH, Gunapala N, Kennedy C (2001) Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and nif mutant strains. Mol Plant Microbe Interact 14:358–366

    Article  CAS  PubMed  Google Scholar 

  • Shi L, Li K, Zhang H et al (2014) Identification of a novel promoter gHp0169 for gene expression in Gluconobacter oxydans. J Biotechnol 175:69–74

    Article  CAS  PubMed  Google Scholar 

  • Sievers M, Swings J (2005) Genus VIII. Gluconacetobacter. In: Brenner DJ, Krieg NR, Staley JT (eds) Bergey’s manual® of systematic bacteriology, 2nd edn. Springer, New York, pp 72–77

  • Spaink HP, Okker RJH, Wijffelman CA et al (1987) Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI. Plant Mol Biol 9:27–39

    Article  CAS  PubMed  Google Scholar 

  • Subashini M, Moushumi Priya A, Sundarakrishnan B, Jayachandran S (2011) Recombinant Gluconacetobacter diazotrophicus containing cry1Ac gene codes for 130-kDa toxin protein. J Mol Microbiol Biotechnol 20:236–242

    Article  CAS  PubMed  Google Scholar 

  • Teixeira KRS, Wülling M, Morgan T et al (1999) Molecular analysis of the chromosomal region encoding the nifA and nifB genes of Acetobacter diazotrophicus. FEMS Microbiol Lett 176:301–309

    Article  CAS  Google Scholar 

  • Tejera NA, Ortega E, Rodés R, Lluch C (2004) Influence of carbon and nitrogen sources on growth, nitrogenase activity, and carbon metabolism of Gluconacetobacter diazotrophicus. Can J Microbiol 50:745–750

    Article  CAS  PubMed  Google Scholar 

  • Wade JT, Grainger DC (2014) Pervasive transcription: illuminating the dark matter of bacterial transcriptomes. Nat Rev Microbiol 12:647–653

    Article  CAS  PubMed  Google Scholar 

  • Wilms I, Overlöper A, Nowrousian M et al (2012) Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens. RNA Biol 9:446–457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors acknowledge Luc Rouws for critical revision of this manuscript, Janaína Rouws and Daniel Ferreira for statistical considerations in constructing the promoter library, and Emanuel M. de Souza for kindly providing vector pPW452. This work has been funded by The National Council for Scientific and Technological Development (CNPq—“National Institute of Science and Technology of Biological Nitrogen Fixation”, grant nº 573.828/2008-3), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Faperj—A.A.L.B. “Iniciação Científica” fellowship, nº E-26/200.930/2015, and grant “Desenvolvimento Científico e Tecnológico Regional”, nº E-26/110.235/2011), and Brazilian Agricultural Research Corporation (Embrapa—Infoseg nº 03.07.09.026.00.00).

Author information

Authors and Affiliations

  1. Embrapa Agrobiologia, Rodovia BR 465 km 7, Seropédica, RJ, Brazil

    Stefan Schwab, Cristiane Alves Pessoa, Amanda Aparecida de Lima Bergami, Nathália Lima de Azevedo Figueiredo, Kátia Regina dos Santos Teixeira & José Ivo Baldani

  2. Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ, Brazil

    Cristiane Alves Pessoa

  3. Instituto de Tecnologia, Universidade Federal Rural do Rio de Janeiro, Rodovia BR 465 km 7, Seropédica, RJ, Brazil

    Amanda Aparecida de Lima Bergami

  4. Instituto de Agronomia, Universidade Federal Rural do Rio de Janeiro, Rodovia BR 465 km 7, Seropédica, RJ, Brazil

    Nathália Lima de Azevedo Figueiredo

Authors
  1. Stefan Schwab
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cristiane Alves Pessoa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amanda Aparecida de Lima Bergami
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nathália Lima de Azevedo Figueiredo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kátia Regina dos Santos Teixeira
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. José Ivo Baldani
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stefan Schwab.

Additional information

Communicated by Jorge Membrillo-Hernández.

Kátia Regina dos Santos Teixeira: In memoriam.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schwab, S., Pessoa, C.A., de Lima Bergami, A.A. et al. Isolation and characterization of active promoters from Gluconacetobacter diazotrophicus strain PAL5 using a promoter-trapping plasmid. Arch Microbiol 198, 445–458 (2016). https://doi.org/10.1007/s00203-016-1203-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-016-1203-y

Keywords

Search

Navigation