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Isolation of transposon mutants and characterization of genes involved in biofilm formation by Pseudomonas fluorescens TC222

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Abstract

Biofilm formation mutants are often found to have defective or altered motility. The motility phenotype was exploited to identify Pseudomonas fluorescens biofilm formation mutants. Fourteen motility mutants were obtained from P. fluorescens isolate TC222 and eight stable mutants were studied further. The eight transposon insertion mutants showed altered ability to form biofilm compared with the parent. Five Tn5-inserted genes from these mutants were cloned and sequenced. Genetic analysis showed that two insertions were located in genes affecting multiple cell surface characteristics, including lipopolysaccharide (rfbD) and polar flagella (fliR). Three genes encoding for a putative Mig-14 family protein (epsB), a probable bacteriophage signal peptide protein (bspA) and a soluble pyridine nucleotide transhydrogenase (pyrA) were reported for the first time to be involved in biofilm formation. Complementation experiments of rfbD and epsB genes proved that biofilm formation of the corresponding mutants could be restored. Further semi-quantitative reverse transcription-PCR analysis showed that both rfbD and epsB can express their transcripts much higher in the complemented strains than that in wild-type strains. The transcripts of both genes in their mutants could not be detected.

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Abbreviations

LPS:

Lipopolysaccharide

NBY:

Nutrient broth containing yeast extract medium

ORFs:

Open reading frames

STH:

Soluble pyridine nucleotide transhydrogenase

SDS:

Sodium dodecylsulfate

SQRT-PCR:

Semi-quantitative reverse transcription-PCR

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Acknowledgment

We thank Dr Søren Molin for kindly providing the plasmid pJBA28. This work was supported by National Significant Basic Research Project Program (2003CB114201 and 2001CB109005). Special thanks to Ms. Wendy C Kain (Cornell University) for revising this manuscript.

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Authors and Affiliations

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100094, People’s Republic of China

    Hongjuan Nian, Jie Zhang, Fuping Song & Liqiang Fan

  2. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, No. 12, South Street, Zhongguancun, Haidian District, Beijing, 100081, People’s Republic of China

    Hongjuan Nian & Dafang Huang

Authors
  1. Hongjuan Nian
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  2. Jie Zhang
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  3. Fuping Song
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  4. Liqiang Fan
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  5. Dafang Huang
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Correspondence to Dafang Huang.

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Communicated by Sebastian Suerbaum.

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Nian, H., Zhang, J., Song, F. et al. Isolation of transposon mutants and characterization of genes involved in biofilm formation by Pseudomonas fluorescens TC222. Arch Microbiol 188, 205–213 (2007). https://doi.org/10.1007/s00203-007-0235-8

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  • DOI: https://doi.org/10.1007/s00203-007-0235-8

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