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1.
Fig. 7

Fig. 7. From: Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant.

Growth of bacterial cultures of WT, crp mutant and its complemented strain (Comp) in M. bovis BCG with supplemented medium. (A) Serine biosynthesis pathway in E. coli. (B) Growth curves of M. bovis BCG WT and crp mutant in different media. Data shown are representative of two biological repeats. The complemented strain is not distinguishable from WT and is not shown in this panel. (C) Growth of M. bovis BCG WT, crp mutant and its complemented strain in different media for 7 d. Data shown are means of three experimental repeats. Error bars denote SD. *, p<0.05; ** p<0.01; ***, p<0.001.

Guangchun Bai, et al. Mol Microbiol. ;82(1):180-198.
2.
FIG. 6

FIG. 6. From: Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant.

Complementation of E. coli serC mutant KL285 with plasmid expressing serC from M. tuberculosis or E. coli. (A and B) PCR confirmation of the presence of serC DNA sequences in the appropriate complemented E. coli strains, using serC primers from M. tuberculosis (A), or E. coli (B). The amplification of E. coli crp was used as a DNA loading control. Lane1, KL285(pMBC664) control; lane 2, KL285(pMBC886) expressing M. tuberculosis serC; lane3, KL285(pMBC887) expressing E. coli serC. (C) Growth of the recombinant strains in the minimal medium with (+ Ser) or without (−Ser) serine. 1, vector-only control strain; 2, M. tuberculosis serC-complemented strain; 3, E. coli serC-complemented strain.

Guangchun Bai, et al. Mol Microbiol. ;82(1):180-198.
3.
Fig. 8

Fig. 8. From: Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant.

Constitutive expression of serC in M. bovis BCG (A and C) and M. tuberculosis (B and D) to address the growth defects caused by crp deletion. (A and B) Comparison of growth among WT, the crp mutant, and each harboring either the promoterless vector control (pMBC283) or vector expressing serC under control of the M. tuberculosis tuf promoter (pMBC1091). Colonies were imaged after 31 d of incubation at 37°C. (C and D) Growth curves of WT, crp mutants, and recombinants expressing of serC under tuf promoter. Data shown are means of three independently repeated experiments at each time point. Error bars denote SD.

Guangchun Bai, et al. Mol Microbiol. ;82(1):180-198.
4.
FIG. 2

FIG. 2. From: Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant.

Transcriptional activity of serC and Rv0885 promoter truncations in M. bovis BCG. (A) Schematic of serC and Rv0885 promoter truncations used in lacZ reporter constructs. The ‘Long’ fragment was truncated from the start of the 16 bp motif, while the ‘Short’ fragment was truncated from the end of the 16 bp motif. The CRPMt binding site is indicated with filled bars. (B) Expression from serC and Rv0885 promoter truncations compared with expression from their respective full length WT sequences using transcriptional lacZ reporter fusions. Activity/OD refers to the amount of beta-galactosidase activity measured in arbitrary units using C2FDG as a substrate per OD650 unit of each bacterial culture. Data are presented as mean values ± standard deviations from three independently repeated biological experiments. *, p<0.05, **, p<0.01 and ***, p<0.001.

Guangchun Bai, et al. Mol Microbiol. ;82(1):180-198.
5.
FIG. 1

FIG. 1. From: Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant.

Transcriptional initiation sites of serC and Rv0885. (A–C) Mapping of the 5′ end of Rv0885 with KM1335 (A), and the 5′ end of serC with KM1609 (B) and KM1608 (C) by primer extension. (D) DNA sequence of serC-Rv0885 intergenic region (top strand reads as sense strand for serC and antisense strand for Rv0885). The TSS for Rv0885 mapped with Rv1335 is indicated with #. The possible TSSs for serC associated with the dominant bands are indicated with * (mapped with KM1609) and with @ (mapped with KM1608), while ● marks start points that correlate with the additional minor extension product for each primer in the area of the Rv0885 start site. The putative −10 region −10) for each gene is underlined. The translation start codons are shown in boldface. The CRPMt binding motif and the motif substitution controls 1 and 2 used in later experiments (‘Sub Ctl 1’ and ‘Sub Ctl 2’) are indicated with boxes for reference.

Guangchun Bai, et al. Mol Microbiol. ;82(1):180-198.
6.
Fig. 9

Fig. 9. From: Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant.

Effects of serC expression during macrophage infection. Macrophages were infected with WT; the newly generated crp deleted M. tuberculosis mutant, crp; the crp mutant carrying a single integrated copy of WT crp, com; an empty expression vector, crp(pMBC283); or a vector that expresses serC from the tuf promoter, crp(pMBC1091). Infection levels were monitored by plating (A) and microscopy (B) at time points ranging from 4 h to 9 d post infection. Only the WT and crp complemented strains showed significant growth within macrophages during this period. The crp mutant failed to grow, but the initial infection level was maintained for the full 9 d assay period, and expression of serC alone did not restore intramacrophage growth in the absence of crp. Data in panel A are the means of two independent experiments, while panel B shows representative images from one of these experiments.

Guangchun Bai, et al. Mol Microbiol. ;82(1):180-198.
7.
FIG. 5

FIG. 5. From: Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant.

Expression of serC and Rv0885 in crp mutants of M. tuberculosis and M. bovis BCG. (A) Schematic of crp mutant construction. Left-side (Left) and right-side (Right) DNA flanking sequences were cloned into a knockout vector on either side of a hygromycin resistance cassette, for homologous recombination with WT strains. Primers used to confirm the junction in mutants are also indicated. (B) Expression of serC and Rv0885 promoter fusions in M. bovis BCG WT and crp mutant measured using lacZ reporter fusions. The promoterless (lac) and tuf promoter-containing constructs were used as null and constitutive expression controls, respectively. Activity/OD refers to the amount of beta-galactosidase activity measured in arbitrary units using C2FDG as a substrate per OD650 unit of each bacterial culture. Error bars denote SD for three repeated experiments; * indicates p<0.05. (C) Regulation of serC and Rv0885 by CRPMt in M. tuberculosis. cDNA was synthesized from M. tuberculosis WT, crp mutant (ko) and its complementary strain (com). The gels shown are representative of three independent experimental repeats. Gels were analyzed using ImageQuant software. The WT/crp mutant ratios of the serC, Rv0885 and Rv0019c are Means ± SEM of three independent experiments, which were normalized by sigA. *, lipQ was only included in one experiment.

Guangchun Bai, et al. Mol Microbiol. ;82(1):180-198.
8.
FIG. 3

FIG. 3. From: Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant.

Substitution of the CRPMt binding motif within the serC and Rv0885 promoters. (A) Schematic of the position where a 16 bp sequence within the serC and Rv0885 promoters was replaced with 16 bp random sequence (open bar). The binding motif core is shown as a filled bar. Controls were replaced at either 18 bp upstream (‘Sub Ctl 1’ in ), or 18 bp downstream (‘Sub Ctl 2’ in ), of the CRPMt binding site. (B) Expression of the serC and Rv0885 promoters with CRP binding motif substitutions in M. bovis BCG, as indicated in panel A. Expression was measured using promoter: lacZ fusions. Activity/OD refers to the amount of beta-galactosidase activity measured in arbitrary units using C2FDG as a substrate per OD650 unit of each bacterial culture. Data are presented as mean values ± standard deviations of three independently repeated biological experiments. *, 0.01<p<0.05. (C) CRPMt binding with the serC-Rv0885 intergenic region and the mutation-containing fragments. The WT and mutation-containing fragments (as shown in panel A) were labeled as probes. CRPMt was used at 0.7, 1.7, 3.5, 7, 17 nM in the EMSA reactions, shown in order from left to right for each group of six lanes in the figure.

Guangchun Bai, et al. Mol Microbiol. ;82(1):180-198.
9.
FIG. 4

FIG. 4. From: Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant.

Point mutation of the CRPMt binding motif within serC and Rv0885 promoters. (A) EMSA showing binding of CRPBCG with full-length native DNA probe from the serC-Rv0885 intergenic region. Lane 1 is probe only; samples in lanes 2 to 7 contain 3.5 nM CRPBCG. Lanes 3 to 7 also include 500-fold excess of unlabeled competitors as follows: 3, 1B5, which is a CRPMt-binding DNA sequence in Mtb that was identified in a previous study (); 4, a 40 bp DNA sequence upstream of the Rv1624c orf that does not contain a CRPMt-binding site, which is used as a negative control; 5, native serC-Rv0885 motif probe; 6, modified G4-to-C; and 7, modified C17-to-G. (B) Schematic showing positions of point mutations within the serC and Rv0885 promoters. Core sequences of the CRPMt binding motif are represented as a filled block, while the position of each mutation is marked with an asterisk. (C) Effect of the point mutations on transcriptional activity of the serC and Rv0885 promoters in M. bovis BCG as measured using lacZ fusions. Activity/OD refers to the amount of beta-galactosidase activity measured in arbitrary units using C2FDG as a substrate per OD650 unit of each bacterial culture. Data are presented as mean values ± standard deviations of three independently repeated biological experiments. **, p<0.01.

Guangchun Bai, et al. Mol Microbiol. ;82(1):180-198.

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