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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 and corresponding microscope images of green fluorescence vs. forward scatter (FSC) of S. wittichii RW1 miniTn5 mutant cells embedded or not in agarose beads and stained with SYTO9. MiniTn5 mutant library as free cells (A,E), empty agarose beads (B,F), agarose beads prepared with a highly concentrated cell culture OD600~1.4 (C,G), or with a diluted cell culture OD600~0.07 (D,H). P4, gate with beads with high cell density; P5, beads with low cell density.
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Figure 1: Flow cytometer diagrams and corresponding microscope images of green fluorescence vs. forward scatter (FSC) of S. wittichii RW1 miniTn5 mutant cells embedded or not in agarose beads and stained with SYTO9. MiniTn5 mutant library as free cells (A,E), empty agarose beads (B,F), agarose beads prepared with a highly concentrated cell culture OD600~1.4 (C,G), or with a diluted cell culture OD600~0.07 (D,H). P4, gate with beads with high cell density; P5, beads with low cell density.

Mentions: Agarose beads containing RW1 mutant cells were analyzed by FC on a FACSAria (BD Biosciences) and using the BD FACSDiva software (version 6.1.3). An aliquot containing the cells-beads solution was stained by adding 1/1000 volume of SYTO9 solution (Invitrogen) and incubating in the dark for 15 min. The stained cell-bead mix was aspirated at approximately 50–100 μl/min and FSC, SSC and green fluorescence (FITC-channel) were recorded. Approximately 900,000 events were detected in the cell-bead initial mix. Gates were set using wild-type RW1 cell suspension (Figure 1A), a suspension of empty beads (Figure 1B), or beads prepared with RW1 cultures with an OD600 of 1.4 (Figure 1C) and 0.07 (Figure 1D). Gate P4 corresponds then to beads carrying a high cell number while P5 includes beads with a low cell density. The presence of free cells (Figure 1E), empty agarose beads (Figure 1F) and cells in beads from gates P4 and P5 (Figures 1G,H, respectively) was confirmed by sorting and subsequent epifluorescence/phase-contrast microscopy. After setting an accurate drop delay value (Accudrop protocol, FACSAria, BD Biosciences, Erembodegem, Belgium), P5 beads were sorted and recovered in a tube (Settings: Voltage FSC 25, SSC 383, FITC 429 / Threshold FSC 1000). The P5 subpopulation was then divided in three fractions. To one of those MM+Km was added (no carbon); to the second MM+Km+SAL (0.5 mM) and to the last one MM+Km+SAL+NaCl was added (to achieve a reduction in water potential of −1.5 MPa). The salicylate concentration (0.5 mM) was lower in this experiment to avoid microcolonies developing too large and escaping the beads. Bead suspensions were incubated at 30°C and 100 rpm for 3 days. A bead sample was analyzed for microcolony growth every day by staining, FC and epifluorescence microscopy. Gates were adjusted for FITC vs. SSC signals: Gate P1 corresponding to beads containing developed microcolonies (high fluorescence) and gate P2 corresponding to beads containing non-developed microcolonies (low fluorescence). Beads, which after 3 days of incubation entered in the P2-gate, were again sorted out individually and placed as microdroplets directly on MM+SAL+Km agar plates. Plates were incubated at 30°C until regular RW1 colonies were visible (~7 days). Transposon mutant colonies were then verified in liquid culture to determine growth rates and biomass yield in presence or absence of NaCl at −1.5 MPa.


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 and corresponding microscope images of green fluorescence vs. forward scatter (FSC) of S. wittichii RW1 miniTn5 mutant cells embedded or not in agarose beads and stained with SYTO9. MiniTn5 mutant library as free cells (A,E), empty agarose beads (B,F), agarose beads prepared with a highly concentrated cell culture OD600~1.4 (C,G), or with a diluted cell culture OD600~0.07 (D,H). P4, gate with beads with high cell density; P5, beads with low cell density.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Flow cytometer diagrams and corresponding microscope images of green fluorescence vs. forward scatter (FSC) of S. wittichii RW1 miniTn5 mutant cells embedded or not in agarose beads and stained with SYTO9. MiniTn5 mutant library as free cells (A,E), empty agarose beads (B,F), agarose beads prepared with a highly concentrated cell culture OD600~1.4 (C,G), or with a diluted cell culture OD600~0.07 (D,H). P4, gate with beads with high cell density; P5, beads with low cell density.
Mentions: Agarose beads containing RW1 mutant cells were analyzed by FC on a FACSAria (BD Biosciences) and using the BD FACSDiva software (version 6.1.3). An aliquot containing the cells-beads solution was stained by adding 1/1000 volume of SYTO9 solution (Invitrogen) and incubating in the dark for 15 min. The stained cell-bead mix was aspirated at approximately 50–100 μl/min and FSC, SSC and green fluorescence (FITC-channel) were recorded. Approximately 900,000 events were detected in the cell-bead initial mix. Gates were set using wild-type RW1 cell suspension (Figure 1A), a suspension of empty beads (Figure 1B), or beads prepared with RW1 cultures with an OD600 of 1.4 (Figure 1C) and 0.07 (Figure 1D). Gate P4 corresponds then to beads carrying a high cell number while P5 includes beads with a low cell density. The presence of free cells (Figure 1E), empty agarose beads (Figure 1F) and cells in beads from gates P4 and P5 (Figures 1G,H, respectively) was confirmed by sorting and subsequent epifluorescence/phase-contrast microscopy. After setting an accurate drop delay value (Accudrop protocol, FACSAria, BD Biosciences, Erembodegem, Belgium), P5 beads were sorted and recovered in a tube (Settings: Voltage FSC 25, SSC 383, FITC 429 / Threshold FSC 1000). The P5 subpopulation was then divided in three fractions. To one of those MM+Km was added (no carbon); to the second MM+Km+SAL (0.5 mM) and to the last one MM+Km+SAL+NaCl was added (to achieve a reduction in water potential of −1.5 MPa). The salicylate concentration (0.5 mM) was lower in this experiment to avoid microcolonies developing too large and escaping the beads. Bead suspensions were incubated at 30°C and 100 rpm for 3 days. A bead sample was analyzed for microcolony growth every day by staining, FC and epifluorescence microscopy. Gates were adjusted for FITC vs. SSC signals: Gate P1 corresponding to beads containing developed microcolonies (high fluorescence) and gate P2 corresponding to beads containing non-developed microcolonies (low fluorescence). Beads, which after 3 days of incubation entered in the P2-gate, were again sorted out individually and placed as microdroplets directly on MM+SAL+Km agar plates. Plates were incubated at 30°C until regular RW1 colonies were visible (~7 days). Transposon mutant colonies were then verified in liquid culture to determine growth rates and biomass yield in presence or absence of NaCl at −1.5 MPa.

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