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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

Culture-density normalized eGFP values in selected RW1 mutants with consistent induction in medium with lowered water potential as a consequence of NaCl-amendment compared to control medium conditions without lower water potential (MM+SAL+Km). Measurements show values after 4 and 8 h of exposure.
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Figure 8: Culture-density normalized eGFP values in selected RW1 mutants with consistent induction in medium with lowered water potential as a consequence of NaCl-amendment compared to control medium conditions without lower water potential (MM+SAL+Km). Measurements show values after 4 and 8 h of exposure.

Mentions: The FC flow diagram of the RW1 mutant library showed that the library contains both cells with a low and a high fluorescence (Figure 2D). The cells falling into the P2 gate were discarded since we assumed they include mostly constitutively eGFP-producing clones. The cells in the P1 gate were sorted out and used to expose to growth conditions of lowered water potential by addition of NaCl. After two rounds of NaCl exposure and fluorescence-assisted sorting of potentially induced cells from P2, some 7000 cells were recovered from P2 and deposited on agar plates for culturing. 768 colonies were picked and rescreened in 96-well microtiter plate format to measure growth and fluorescence in the presence of NaCl compared to control conditions. A total of 45 mutant strains displayed a culture-density normalized eGFP signal 1.3–2 times higher when exposed to NaCl than in control media. After repeated verification, 16 of the 45 clones showed consistent higher normalized eGFP fluorescence when exposed to NaCl compared to the control (Figure 8). In some of those clones the signals developed only after 4 h and in others after 8 h of NaCl-exposure. On average, normalized eGFP signals in NaCl-induced cultures were between 1.3 and 1.6 times higher than in the control (Table 1).


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)

Culture-density normalized eGFP values in selected RW1 mutants with consistent induction in medium with lowered water potential as a consequence of NaCl-amendment compared to control medium conditions without lower water potential (MM+SAL+Km). Measurements show values after 4 and 8 h of exposure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Culture-density normalized eGFP values in selected RW1 mutants with consistent induction in medium with lowered water potential as a consequence of NaCl-amendment compared to control medium conditions without lower water potential (MM+SAL+Km). Measurements show values after 4 and 8 h of exposure.
Mentions: The FC flow diagram of the RW1 mutant library showed that the library contains both cells with a low and a high fluorescence (Figure 2D). The cells falling into the P2 gate were discarded since we assumed they include mostly constitutively eGFP-producing clones. The cells in the P1 gate were sorted out and used to expose to growth conditions of lowered water potential by addition of NaCl. After two rounds of NaCl exposure and fluorescence-assisted sorting of potentially induced cells from P2, some 7000 cells were recovered from P2 and deposited on agar plates for culturing. 768 colonies were picked and rescreened in 96-well microtiter plate format to measure growth and fluorescence in the presence of NaCl compared to control conditions. A total of 45 mutant strains displayed a culture-density normalized eGFP signal 1.3–2 times higher when exposed to NaCl than in control media. After repeated verification, 16 of the 45 clones showed consistent higher normalized eGFP fluorescence when exposed to NaCl compared to the control (Figure 8). In some of those clones the signals developed only after 4 h and in others after 8 h of NaCl-exposure. On average, normalized eGFP signals in NaCl-induced cultures were between 1.3 and 1.6 times higher than in the control (Table 1).

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