Journal: Nucleic Acids Research

Article Title: Characterization of the MgsR-dependent promoter structure in Bacillus subtilis —application of a novel pHIS plasmid-based screening system for promoter element analysis

doi: 10.1093/nar/gkaf636

Figure Lengend Snippet: Identification of the MgsR operator by the pHIS plasmid-based screening system. ( A ) Schematic representation of the ydbD promoter upstream region, containing the native ydbD promoter (top, MHP01, highlighted in green) and the versions with a deleted (middle, MHP02, highlighted in gray) and a substituted MgsR operator (bottom, MHP03, highlighted in orange), located in front of the read-reporter gene gfp . Underlined sequences (middle) depict a potential MgsR operator, originating from the fusion with the downstream flanking region. ( B ) Northern blot experiments displaying the gfp expression patterns of the three pHIS constructs (native MgsR operator, deleted and substituted variant) and ( C ) bar plots representing quantitative data from at least three biological replicates and showing normalized GFP fluorescence intensities of the in vivo analysis in a B. subtilis wild type and its isogenic Δ mgsR mutant with their respective standard deviation. Statistical significance in the bar plots is denoted adjusted by ** P -values ≤.01. Cells were grown in SMM and as soon as an optical density of OD 500nm = 0.4 was reached, 4% (v/v) ethanol was added to the cultures. Native ydbD promoter in the respective B. subtilis strains is shown in a green color palette, the deleted MgsR operator in a gray color palette, and the substituted MgsR operator in an orange color palette. Equal loading of northern blots was verified by methylene blue staining (see ).

Article Snippet: MHP00 , Native ydbD promoter, intact MgsR operator , None.

Techniques: Plasmid Preparation, Northern Blot, Expressing, Construct, Variant Assay, Fluorescence, In Vivo, Mutagenesis, Standard Deviation, Staining

Journal: Nucleic Acids Research

Article Title: Characterization of the MgsR-dependent promoter structure in Bacillus subtilis —application of a novel pHIS plasmid-based screening system for promoter element analysis

doi: 10.1093/nar/gkaf636

Figure Lengend Snippet: Ectopic Integration of the MgsR operator confers MgsR-dependent regulation to the non-target SigB-type promoter yocB . ( A ) Schematic representation of the yocB promoter upstream region (MHP08) and the ydbD / yocB promoter hybrid (MHP10), located in front of the read-reporter gene gfp gene. The MgsR operator is highlighted in green. ( B ) Northern blot experiments displaying the gfp expression patterns of the two pHIS constructs (native yocB promoter and ydbD / yocB hybrid promoter) and ( C ) bar plots representing quantitative data from at least three biological replicates and showing normalized GFP fluorescence intensities of the in vivo analysis in a B. subtilis wild type and its isogenic Δ sigB and Δ mgsR mutants with their respective standard deviation. Statistical significance in the bar plots is denoted adjusted by ** P -values ≤.01 and *** P -values ≤.001. Cells were grown in SMM and as soon as an optical density of OD 500nm = 0.4 was reached, 4% (v/v) ethanol was added to the cultures. Native yocB promoter in the respective B. subtilis strains is shown in a gray color palette, and the ydbD / yocB promoter hybrid in a green color palette. Equal loading of northern blots was verified by methylene blue staining (see ).

Article Snippet: MHP00 , Native ydbD promoter, intact MgsR operator , None.

Techniques: Northern Blot, Expressing, Construct, Fluorescence, In Vivo, Standard Deviation, Staining

Journal: Nucleic Acids Research

Article Title: Characterization of the MgsR-dependent promoter structure in Bacillus subtilis —application of a novel pHIS plasmid-based screening system for promoter element analysis

doi: 10.1093/nar/gkaf636

Figure Lengend Snippet: Identification of a −35-like silencing element affecting transcriptional efficiency. ( A ) Schematic representation of the ydbD / yocB promoter hybrid (MHP10, green) and substitution of the −35-like region (MHP12, orange), located in front of the reporter gene gfp gene. The −35-like region is underscored, while the substituted version is highlighted in orange. ( B ) Northern blot experiments displaying the gfp expression patterns of the two pHIS constructs ( ydbD / yocB promoter hybrid with or without the −35-like region) and ( C ) bar plots representing quantitative data from at least three biological replicates and showing normalized GFP fluorescence intensities of the in vivo analysis in a B. subtilis wild type and its isogenic Δ sigB and Δ mgsR mutants with their respective standard deviation. Statistical significance in the bar plots is denoted by adjusted ** P -values ≤.01 and * P -values ≤.05. Cells were grown in SMM and as soon as an optical density of OD 500nm = 0.4 was reached, 4% (v/v) ethanol was added to the cultures. Native yocB promoter in the respective B. subtilis strains is shown in a gray color palette, and the ydbD / yocB promoter hybrid in a green color palette. Equal loading of northern blots was verified by methylene blue staining (see ).

Article Snippet: MHP00 , Native ydbD promoter, intact MgsR operator , None.

Techniques: Northern Blot, Expressing, Construct, Fluorescence, In Vivo, Standard Deviation, Staining

Journal: Nucleic Acids Research

Article Title: Characterization of the MgsR-dependent promoter structure in Bacillus subtilis —application of a novel pHIS plasmid-based screening system for promoter element analysis

doi: 10.1093/nar/gkaf636

Figure Lengend Snippet: Identifying the putative MgsR operator. ( A ) Northern blot experiments displaying the expression profiles of chromosomal ydbD , yocB , and yraA under control conditions and 10 min after 4% (v/v) ethanol stress in different B. subtilis strains (BSB1 WT, Δ sigB , Δ mgsR , Δ spx , Δ mgsR + Δ spx ). Cells were grown in SMM to an OD 500nm = 0.4 and then ethanol was added. ( B ) Multiple sequence alignment of upstream regions of genes significantly regulated by MgsR is shown . Putative MgsR operator highlighted by a dark box. Sequence logo was generated (weblogo.berkeley.edu) with the multiple sequence alignment to display conserved bases at distinct positions. Equal loading of northern blots was verified by methylene blue staining (see ).

Article Snippet: MHP00 , Native ydbD promoter, intact MgsR operator , None.

Techniques: Northern Blot, Expressing, Control, Sequencing, Generated, Staining