Article Figures & Data
Figures
- FIG. 1.
Partial sequence alignment of YvcJ homologues in various bacteria, including Bacillus anthracis, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae, as well as B. subtilis and E. coli. The putative Walker A and B motifs are indicated by the two shaded boxes. Walker A is normally quoted in the literature by the consensus sequence (A/G)X4GK(T/S), which is centered at a loop between a β-strand and an α-helix (35, 39). The Walker B motif, much less conserved, can be represented by the consensus sequence DX2G (38), located at the end of a hydrophobic β-sheet.
- FIG. 2.
YvcJ is a nucleotide-binding protein. (A) Coomassie blue-stained SDS-PAGE gel showing a partial proteolysis profile of YvcJ. YvcJ was incubated with endoproteinase Glu-C (Promega) in the absence or presence of ATP or GTP for 0, 5, 15, or 30 min at 37°C. The digestion profiles were assessed by 15% SDS-PAGE. (B) Effect of Mant-ATP on the fluorescence of YvcJ. Increasing concentrations of Mant-ATP (from 0 μM to 40 μM) were added to a 2-ml assay medium containing 25 mM HEPES-KOH (pH 8.0), 1 mM MgCl2, and 0.5 μM YvcJ, and the fluorescence intensity was recorded after each addition. The FRET, taken as the increase in fluorescence between 400 and 500 nm, was plotted against the concentration of Mant-ATP. A.U., arbitrary units.
- FIG. 3.
Growth curves (A) and transformation frequencies (B) of wild-type and yvcJ mutant strains in which ComK was overexpressed. Wild-type strain 168 (squares) and mutant strains SG91 (diamonds), SG119 (circles), and SG120 (triangles) were grown on MD medium. comK expression was induced by 1 mM IPTG at an optical density at 600 nm of 0.7 (first arrow), and cells were transformed 2 h afterwards (second arrow) by using 0.5 μg of a chromosomal DNA carrying a kanamycin marker. Transformation frequencies were determined by selection for Kmr. The transformation frequency corresponds to the ratio between the number of transformants per milliliter and the number of cells per milliliter.
- FIG. 4.
Microscopy of cells harboring comK-gfp in strains SG147 (A) and SG152 (with yvcJ deleted) (B). Shown are images of cells from the cultures of strains SG147 and SG152 90 min after the transition between the exponential and the stationary-growth phase. (Left) Phase-contrast microscopy; (right) fluorescence microscopy.
- FIG. 5.
ATP-dependent phosphorylation in the presence of YvcJ. Wild-type strain 168 (lanes 1 and 2) and strains BD1243 (lanes 3 and 4) and BD3836 (lanes 5 and 6) were grown on MD medium, and cells were collected at the onset of the stationary phase. For strain BD3836, comK overexpression was induced by addition of 1 mM IPTG to the growth medium. Crude extracts were prepared and then phosphorylated using [γ-33P]ATP in the absence (lanes 1, 3, and 5) or the presence (lanes 2, 4, and 6) of purified YvcJ. Phosphorylation assays were analyzed by 15% SDS-PAGE. Gels were then dried and exposed to autoradiography.
Tables
- TABLE 1.
B. subtilis strains used in this study
Strain Genotype or description Source or reference 168 trpC2 Laboratory stock SG91 trpC2 ΔyvcJ::cat This work SG106 trpC2 ΔyvcJ::cat amyE::yvcI kan This work SG107 trpC2 ΔyvcJ::cat amyE::yvcIJ kan This work BD3836 hisB2 leu-8 metB5 amyE::P hs -comK spec 27 SG119 trpC2 amyE::P hs -comK spec BD3836→168 SG120 trpC2 ΔyvcJ::cat amyE::P hs -comK spec SG119→SG91 BD2528 hisB2 leu-8 metB5 pUB110 comS kan 15 SG121 trpC2 pUB110 comS kan BD2528→168 SG122 trpC2 ΔyvcJ::cat pUB110 comS kan SG121→SG91 SG131 trpC2 ΔyvcJ::tet This work BD3196 hisB2 leu-8 metB5 Δrok::kan 1 BD2711 hisB2 leu-8 metB5 comK-gfp cat 37 SG147 hisB2 leu-8 metB5 Δrok::kan comK-gfp cat BD2711→BD3196 SG152 hisB2 leu-8 metB5 ΔyvcJ::tet Δrok::kan PcomK-gfp cat SG131→SG147 BD1243 hisAl leuA8 metB5 comA124::Tn9171acZ 33 - TABLE 2.
Relative expression levels of glmS and of genes involved in the competence pathway in a yvcJ mutant strain from transcriptome analysis
Gene Expression level Description glmS 1.39 Glucosamine-6-phosphate synthase Genes missing in the microarray addA ATP-dependent DNase comEC Late competence operon required for DNA binding and uptake comP Two-component sensor histidine kinase involved in early competence Genes with unchanged expression comA 0.84 Two-component response regulator of late competence genes and surfactin production comX 1.00 Competence pheromone precursor comQ 1.16 Transcriptional regulator of late competence operon and surfactin expression comS 0.71 Assembly link between regulatory components of the competence pathway srfAA 0.85 Surfactin synthetase/competence pnpA 0.85 Polynucleotide phosphorylase (PNPase) ylbF 0.73 Unknown; positively controls ComK at a posttranscriptional level mecA 1.27 Negative regulator of competence clpX 1.13 ATP-dependent Clp protease clpC 0.84 Class III stress response-related ATPase clpP 0.80 ATP-dependent Clp protease proteolytic subunit ypbH 1.18 Unknown; similar to negative regulator of competence; MecA homologue codY 0.88 Transcriptional pleiotropic repressor degU 0.92 Two-component response regulator involved in degradative enzyme and competence abrB 0.81 Transcriptional pleiotropic regulator of transition state genes rapC 1.01 Response regulator, aspartate phosphatase addB 0.97 ATP-dependent DNase rok 1.29 Repressor of comK recA 0.67 Multifunctional protein involved in homologous recombination and DNA repair spx 1.70 Negative effector of competence spo0A 1.35 Two-component response regulator central for the initiation of sporulation Genes repressed at least twofold in the yvcJ mutant strain comK 0.42 Competence transcription factor comGA 0.23 Late competence gene comGB 0.24 DNA transport machinery comGC 0.27 Exogenous DNA binding comGD 0.26 DNA transport machinery comGE 0.27 DNA transport machinery comGF 0.29 DNA transport machinery comGG 0.29 DNA transport machinery comER 0.29 Nonessential gene for competence comEA 0.20 Exogenous DNA-binding protein comEB 0.39 Nonessential gene for competence comFA 0.21 Late competence protein required for DNA uptake comFB 0.26 Late competence gene comFC 0.28 Late competence gene ywpH 0.26 Unknown; similar to single-strand DNA-binding protein nin 0.34 Inhibitor of the DNA-degrading activity of NucA comC 0.41 Late competence protein yvyF 0.36 Unknown; similar to flagellar protein nucA 0.33 Membrane-associated nuclease - TABLE 3.
ComS overexpression bypasses yvcJ deletion for competence and gene expressiona
Strain Relevant genotype Transformation frequency (106) Relative gene expression determined by q-RT-PCR comS comK comGA 168 Wild type 46.0 ± 7.1 1 1 1 SG91 yvcJ 6.6 ± 1.6 0.7 ± 0.3 0.60 ± 0.18 0.60 ± 0.21 SG121 pcomS 64.5 ± 10.6 3.1 ± 0.4 1.06 ± 0.03 1.27 ± 0.06 SG122 yvcJ pcomS 31.5 ± 7.8 4.9 ± 1.8 1.60 ± 0.39 1.65 ± 0.44 ↵ a Cells of the wild-type strain and of yvcJ mutant strains in which ComS was overexpressed were transformed with 250 ng of a chromosomal DNA containing a spectinomycin cassette. Transformation frequencies were determined as described in Materials and Methods. The relative expression of the comS, comK, and comGA genes in the wild-type strain and in mutant strains in which ComS was overexpressed was determined by q-RT-PCR. All the values are averages of data from at least three independent experiments.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Table S1, primers; Table S2, comparison of expression of genes involved in the competence pathway.
MS Word document, 57K.
- Supplemental file 1
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Table S1, primers; Table S2, comparison of expression of genes involved in the competence pathway.
