Article Figures & Data
Figures
- FIG 1
Relative expression of selected genes of transcriptional regulators positively regulated by BvgA~P. Values are shown as fold difference using the 2−ΔΔCT method (65). Error bars indicate standard deviations among three independent biological replicates for each condition with more than two different trials. Blue bars, FD of WT without MgSO4 versus WT with MgSO4; red bars, FD of WT without MgSO4 versus ΔbvgAS strain without MgSO4. Student’s t test was used for statistical analysis, and genes showing significant differences between WT without MgSO4 and WT with MgSO4 or between WT without MgSO4 and ΔbvgAS strain without MgSO4 are labeled as *** (P < 0.001), ** (P < 0.01), or * (P < 0.05). “ns” represents statistically nonsignificant.
- FIG 2
Relative expression of selected genes involved in metabolic pathways negatively affected by BvgA~P, as measured by RT-qPCR. Values are shown as fold difference using the 2−ΔΔCT method (65). Error bars indicate standard deviations among three independent biological replicates for each condition with more than two different trials. Blue bars, FD of WT without MgSO4 versus WT with MgSO4; red bars, FD of WT without MgSO4 versus ΔbvgAS strain without MgSO4. Bracketed genes are located in the same locus of the genome. Student’s t test was used for statistical analysis, and genes showing significant differences between WT without MgSO4 and WT with MgSO4 or between WT without MgSO4 and ΔbvgAS strain without MgSO4 are labeled as *** (P < 0.001), ** (P < 0.01), or * (P < 0.05).
- FIG 3
PbrpL is directly activated by BvgA~P. (A) Denaturing polyacrylamide gels showing RNA obtained after in vitro transcription of the indicated templates using (as indicated) B. pertussis RNAP alone (−), RNAP plus BvgA (+), or RNAP plus BvgA~P (P). The expected transcripts (nucleotides) are indicated. (B) Primer extension analysis of in vitro RNA to identify the start site of PbrpL. A denaturing gel of primer extension products is shown. Left lanes are sequencing ladders; right lanes show primer extension products for RNA obtained in the absence of BvgA (−), the presence of BvgA~P (P), or the presence of nonphosphorylated BvgA (+). (C) Sequence of the brpL promoter, determined from primer extension analyses in panel A, and of the promoters for BP1005 and BP3441, determined from transcript length in in vitro transcription in panel B. Assigned −10 and −35 elements (BP1005 and BP3441) are in blue; +1 is in red.
- FIG 4
FeBABE cleavage sites for PbrpL and PfhaB. (A) Denaturing gels showing products of FeBABE cleavage using complexes formed with PbrpL (left) or PfhaB (right), 32P end labeled on either the template or the nontemplate strand, B. pertussis RNAP, and (as indicated) either phosphorylated BvgAT194C-FeBABE or phosphorylated BvgAV148C-FeBABE. The G+A lanes correspond to DNA ladders generated by Maxam-Gilbert sequencing with the positions relative to the transcription start sites indicated (36). Arrowheads denote sites of cleavage. (B) Sequences of PbrpL and PfhaB with the cut sites generated by BvgAT194C-FeBABE (pink arrowheads) and by BvgAV148C-FeBABE (purple arrowheads). BvgA binding sites are boxed in orange; the −10 regions are shown and boxed in blue. (An alternative −10 element for PfhaB is indicated in the dashed purple box.) Transcription start sites (+1) are in red. Numbers above the inverted red arrows indicate the predicted score for BvgA binding half-sites in each promoter according to the algorithm reported by Merkel et al. (67). For instance, the sequence of the perfect inverted repeat is TAGGAAATTTCCTA, whose score is given as “0.”
Tables
TABLE S1
(A) Entire set of RNA-seq data. (B) Genes positively regulated by BvgA~P. Genes whose expressions have an FD of ≥1.6 with a P value of <0.05 are considered to be significant. Genes in blue are newly identified in this study; the gene in red is not Bvg(+) as indicated by RT-qPCR. Genes highlighted in yellow were assessed by RT-qPCR. 1Possible function identified by PSI-BLAST in this study. 2Possible function assigned in the work of Ahuja et al. (32). A total of 245 genes are identified as Bvg(+), 237 by RNA-seq and 8 from RT-qPCR. One hundred twenty-one genes are newly identified as Bvg(+) in this study. (C) Genes negatively regulated by BvgA~P. Genes whose expressions have an FD of ≥1.6 with a P value of <0.05 are considered to be significant. Genes in blue are newly identified in this study; the gene in red is not Bvg(−) as indicated by RT-qPCR. Genes highlighted in yellow were assessed by RT-qPCR. 1Possible function identified by PSI-BLAST in this study. A total of 326 genes are identified as Bvg(−), 321 by RNA-seq and 5 from RT-qPCR. Two hundred thirty-two genes are newly identified as Bvg(−) in this study. Download TABLE S1, XLSX file, 6.6 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S1
RT-qPCR analyses of genes identified as positively regulated by BvgA~P by the RNA-seq analyses. (A) Well-known vags; (B) T3SS genes; (C) genes of membrane-associated transporter proteins and hypothetical proteins. Values are shown as a fold difference using the 2−ΔΔCT method. Error bars indicate standard deviations among three independent biological replicates for each condition with more than two different trials. Blue bar, FD of WT without MgSO4 versus WT with MgSO4; red bar, FD of WT without MgSO4 versus ΔbvgAS without MgSO4. Download FIG S1, EPS file, 4.7 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S2
RT-qPCR analyses of genes identified as negatively regulated by BvgA~P by the RNA-seq analyses. (A) Well-known vrgs; (B) other vrgs, including genes for cold shock membrane proteins and genes with hypothetical functions. (Cold shock genes are bracketed.) Values are shown as a fold difference using the 2−ΔΔCT method. Error bars indicate standard deviations among three independent biological replicates for each condition with more than two different trials. Blue bar, FD of WT without MgSO4 versus WT with MgSO4; red bar, FD of WT without MgSO4 versus ΔbvgAS without MgSO4. Download FIG S2, EPS file, 3.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S2
Well-known vags, T3SS genes, and genes of membrane-associated transporter proteins and hypothetical proteins. (A) Well-known vags positively regulated by BvgA~P; (B) T3SS genes positively regulated by BvgA~P; (C) genes of membrane-associated transporter proteins and hypothetical proteins positively regulated by BvgA~P. Download TABLE S2, DOCX file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S3
Well-known vrgs and other vrgs, including genes for cold shock and membrane proteins and genes with hypothetical functions. (A) Well-known vrgs; (B) other vrgs, including genes for cold shock and membrane proteins and genes with hypothetical functions. Download TABLE S3, DOCX file, 0.03 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S4
Genes involved in various metabolic pathways negatively regulated by BvgA~P. Download TABLE S4, DOCX file, 0.05 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S5
RT-qPCR analysis for other possible vags and vrgs. Genes were previously identified as vags in the work of Cummings et al. (9) in B. pertussis Tohama I,1 in B. pertussis GMT-1,2 in B. bronchiseptica RB50,3 or in B. bronchiseptica 77.4,5 Genes were previously identified as vrgs in the work of Cummings et al. (9). (A) Possible vag transcriptional regulators, not identified by our RNA-seq; (B) genes reported as vrgs in B. bronchiseptica. Download TABLE S5, DOCX file, 0.02 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S3
Cleavage of PbrpL by FeBABE-conjugated BvgA~P in transcription complexes made with E. coli RNAP. (A) Portion of denaturing polyacrylamide gels showing PbrpL RNA (left) or PfhaB RNA (right) obtained after in vitro transcription using, as indicated, E. coli RNAP alone (-), RNAP plus BvgA (+), or RNAP plus BvgA~P (P). (B) Denaturing gels showing products of FeBABE cleavage using complexes formed with PbrpL (left) or PfhaB (right), 32P end labeled on either the template or the nontemplate strand, E. coli RNA polymerase, and, as indicated, either phosphorylated BvgAT194C-FeBABE or phosphorylated BvgAV148C-FeBABE. The G+A lanes correspond to DNA ladders generated by Maxam-Gilbert sequencing with the positions relative to the transcription start sites indicated. Arrowheads denote sites of cleavage. (C) Sequences of PbrpL and PfhaB with the cut sites generated by BvgAT194C-FeBABE (pink arrowheads) and by BvgAV148C-FeBABE (purple arrowheads). BvgA binding sites are boxed in orange; the −10 regions are boxed in blue. (An alternative −10 element for PfhaB is indicated in the dashed purple box.) Transcription start sites (+1) are in red. Numbers above the inverted red arrows indicate the predicted score for BvgA binding half-sites in each promoter according to the algorithm reported by Merkel et al. (67). Download FIG S3, TIF file, 25.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
