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Characterization of multiple promoters and transcript stability in the sacB–sacC gene cluster in Zymomonas mobilis

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Abstract

In Zymomonas mobilis, the extracellular levansucrase (SacB) and extracellular sucrase (SacC) are involved in sucrose hydrolysis. Genes coding for these two enzymes (sacB and sacC) are arranged in a cluster in the genome and separated by a short intervening sequence. The level of sacC transcript was 12-fold higher than that of sacB transcript. On the other hand, transcript stability analysis in sucrose grown cultures revealed that the half-life of the sacB transcripts (153 s) was more than twofold higher than that of sacC transcript (66 s). The decay curves of sacB and sacC transcripts analyzed by the semi-quantitative RT-PCR correlated well with the decay curves of the respective enzyme activities. In the sacB promoter disruption mutant, Z. moblis BT2, the extracellular sucrase activity decreased from 2.6 to 2.0 U mg−1 in sucrose medium due to the loss of SacB expression. The expression of sacC in the absence of the sacB promoter suggested that these two genes could be transcribed as different mRNAs. The promoter-lacZ fusion studies in Escherichia coli proved that the short intervening region acts as a strong promoter for the sacC gene.

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Abbreviations

MU:

Miller units

OD600 :

Optical density at 600 nm

RMG:

Rich medium with glucose

SQ-RT-PCR:

Semi-quantitative reverse transcription PCR

PsacB :

sacB promoter

PsacC :

sacC promoter

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Acknowledgments

The authors gratefully thank Department of Science and Technology (Indo-UK project: INT/UK/P-8/99) and Department of Biotechnology (BT/PR3445/AGR/16/283/2002-III), New Delhi, India for the research grants as well as Prof. K. T. Shanmugam, University of Florida, Florida, USA for generously providing the E. coli strains and phages used in this study. The central facility at the Centre for Excellence in Genomic Sciences, Madurai Kamaraj University is gratefully acknowledged.

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Author notes

  1. V. Senthilkumar

    Present address: Biochemistry building, Faculty of Natural Sciences, Imperial College London, Exhibition Rd, South Kensington, London, SW7 2AZ, UK

Authors and Affiliations

  1. Department of Genetics, Center for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625021, India

    V. Senthilkumar, J. Rajendhran & P. Gunasekaran

  2. Department of Molecular Microbiology, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

    S. J. W. Busby

Authors
  1. V. Senthilkumar
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  2. J. Rajendhran
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  3. S. J. W. Busby
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  4. P. Gunasekaran
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Corresponding author

Correspondence to P. Gunasekaran.

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Communicated by Jorge Membrillo-Hernández.

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Senthilkumar, V., Rajendhran, J., Busby, S.J.W. et al. Characterization of multiple promoters and transcript stability in the sacB–sacC gene cluster in Zymomonas mobilis . Arch Microbiol 191, 529–541 (2009). https://doi.org/10.1007/s00203-009-0479-6

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  • DOI: https://doi.org/10.1007/s00203-009-0479-6

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