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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

Flow cytometer diagrams of green fluorescence vs. forward scatter (FSC) of S. wittichii RW1 wild-type cells (A), E. coli BW20767 (pRL27-egfp) (B), RW1 (miniTn5-egfp) with constitutively high eGFP production (C), and the uninduced RW1Tn5-egfp library (D). P1 was used as gate for low fluorescence whereas P2 was used to differentiate cells with high green fluorescence (see further Figure 5 for explanation of the screening procedure).
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Figure 2: Flow cytometer diagrams of green fluorescence vs. forward scatter (FSC) of S. wittichii RW1 wild-type cells (A), E. coli BW20767 (pRL27-egfp) (B), RW1 (miniTn5-egfp) with constitutively high eGFP production (C), and the uninduced RW1Tn5-egfp library (D). P1 was used as gate for low fluorescence whereas P2 was used to differentiate cells with high green fluorescence (see further Figure 5 for explanation of the screening procedure).

Mentions: The RW1 pRL27-egfp library was screened for cells producing a higher eGFP signal under growth conditions with decreased SP compared to the signal in control media. The assumption here was that an increased eGFP production under lower water potential would indicate that the insertion of the transposable element is within or close to a gene higher expressed under solute stress, and thus perhaps implicated in resisting this stress. A 1 ml aliquot of the library mix was taken out of the −80° storage, slowly thawed, diluted in 50 ml of fresh media (MM+SAL+Km, 5 mM) and incubated overnight at 30°C on a rotary shaker at 180 rpm. Single cell eGFP intensities in the library mutant cultures were determined by FC using the FITC-channel (FACSAria, BD Biosciences). Pure cultures of RW1 and E. coli BW20767 were employed to define the fluorescence level of cells not expressing eGFP (Figures 2A,B, P1 gate). An RW1 transposon mutant recovered from plate showing constitutive eGFP expression was selected to define the high fluorescence gate (Figure 2C, P2 gate).


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)

Flow cytometer diagrams of green fluorescence vs. forward scatter (FSC) of S. wittichii RW1 wild-type cells (A), E. coli BW20767 (pRL27-egfp) (B), RW1 (miniTn5-egfp) with constitutively high eGFP production (C), and the uninduced RW1Tn5-egfp library (D). P1 was used as gate for low fluorescence whereas P2 was used to differentiate cells with high green fluorescence (see further Figure 5 for explanation of the screening procedure).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Flow cytometer diagrams of green fluorescence vs. forward scatter (FSC) of S. wittichii RW1 wild-type cells (A), E. coli BW20767 (pRL27-egfp) (B), RW1 (miniTn5-egfp) with constitutively high eGFP production (C), and the uninduced RW1Tn5-egfp library (D). P1 was used as gate for low fluorescence whereas P2 was used to differentiate cells with high green fluorescence (see further Figure 5 for explanation of the screening procedure).
Mentions: The RW1 pRL27-egfp library was screened for cells producing a higher eGFP signal under growth conditions with decreased SP compared to the signal in control media. The assumption here was that an increased eGFP production under lower water potential would indicate that the insertion of the transposable element is within or close to a gene higher expressed under solute stress, and thus perhaps implicated in resisting this stress. A 1 ml aliquot of the library mix was taken out of the −80° storage, slowly thawed, diluted in 50 ml of fresh media (MM+SAL+Km, 5 mM) and incubated overnight at 30°C on a rotary shaker at 180 rpm. Single cell eGFP intensities in the library mutant cultures were determined by FC using the FITC-channel (FACSAria, BD Biosciences). Pure cultures of RW1 and E. coli BW20767 were employed to define the fluorescence level of cells not expressing eGFP (Figures 2A,B, P1 gate). An RW1 transposon mutant recovered from plate showing constitutive eGFP expression was selected to define the high fluorescence gate (Figure 2C, P2 gate).

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