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Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery.

Coronado E, Roggo C, van der Meer JR - Front Microbiol (2014)

Bottom Line: Conditions of low water potential were mimicked by adding NaCl to the growth media.Three different mutant selection or separation method were tested which, however recovered different mutants.Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Microbiology, University of Lausanne Lausanne, Switzerland.

ABSTRACT
The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

No MeSH data available.


Related in: MedlinePlus

Schematic diagram of the flow cytometry screening procedure followed to detect RW1 transposon mutants with an increased fluorescence as a result of NaCl addition. The RW1 pRL27-egfp library was first sorted to separate the high fluorescence (P2) from low fluorescence clones (P1). The P1 subpopulation was then exposed to NaCl, incubated during 2 and 6 h and the fluorescence measured. The clones showing a high fluorescence after salt addition were recovered from gate P2 and plated on MM+SAL+Km. Once colonies developed on the plates, they were individually transferred to microtiter plates, where they were kept as master plates.
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Figure 7: Schematic diagram of the flow cytometry screening procedure followed to detect RW1 transposon mutants with an increased fluorescence as a result of NaCl addition. The RW1 pRL27-egfp library was first sorted to separate the high fluorescence (P2) from low fluorescence clones (P1). The P1 subpopulation was then exposed to NaCl, incubated during 2 and 6 h and the fluorescence measured. The clones showing a high fluorescence after salt addition were recovered from gate P2 and plated on MM+SAL+Km. Once colonies developed on the plates, they were individually transferred to microtiter plates, where they were kept as master plates.

Mentions: As an alternative to the traditional replica plating screening, which is a rather long and tedious process, and to the agarose beads screening, which gave us a very low recovery (only one clone consistently had a lower growth in salt media), a third screening method was developed by creating a new transposon mutant library using a modified pRL27 plasposon vector which carried a promoterless egfp. A new transposon library of around 22,000 mutants was produced by conjugation of RW1 and E. coli BW20767 (pRL27::miniTn5-egfp). In this case, the mutant library was screened for an increased eGFP signal in single cells when exposed to media with decreased SP (−1.5 MPa). The procedure followed is depicted in Figure 7.


Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery.

Coronado E, Roggo C, van der Meer JR - Front Microbiol (2014)

Schematic diagram of the flow cytometry screening procedure followed to detect RW1 transposon mutants with an increased fluorescence as a result of NaCl addition. The RW1 pRL27-egfp library was first sorted to separate the high fluorescence (P2) from low fluorescence clones (P1). The P1 subpopulation was then exposed to NaCl, incubated during 2 and 6 h and the fluorescence measured. The clones showing a high fluorescence after salt addition were recovered from gate P2 and plated on MM+SAL+Km. Once colonies developed on the plates, they were individually transferred to microtiter plates, where they were kept as master plates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219479&req=5

Figure 7: Schematic diagram of the flow cytometry screening procedure followed to detect RW1 transposon mutants with an increased fluorescence as a result of NaCl addition. The RW1 pRL27-egfp library was first sorted to separate the high fluorescence (P2) from low fluorescence clones (P1). The P1 subpopulation was then exposed to NaCl, incubated during 2 and 6 h and the fluorescence measured. The clones showing a high fluorescence after salt addition were recovered from gate P2 and plated on MM+SAL+Km. Once colonies developed on the plates, they were individually transferred to microtiter plates, where they were kept as master plates.
Mentions: As an alternative to the traditional replica plating screening, which is a rather long and tedious process, and to the agarose beads screening, which gave us a very low recovery (only one clone consistently had a lower growth in salt media), a third screening method was developed by creating a new transposon mutant library using a modified pRL27 plasposon vector which carried a promoterless egfp. A new transposon library of around 22,000 mutants was produced by conjugation of RW1 and E. coli BW20767 (pRL27::miniTn5-egfp). In this case, the mutant library was screened for an increased eGFP signal in single cells when exposed to media with decreased SP (−1.5 MPa). The procedure followed is depicted in Figure 7.

Bottom Line: Conditions of low water potential were mimicked by adding NaCl to the growth media.Three different mutant selection or separation method were tested which, however recovered different mutants.Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Microbiology, University of Lausanne Lausanne, Switzerland.

ABSTRACT
The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

No MeSH data available.


Related in: MedlinePlus