Article Figures & Data
Figures
- Fig. 1.
Polymorphism at the Bacillus QS locus. DNA sequences of B. subtilis 168 and RS-B-1, and of B. mojavensis RO-H-1 and of B. natto NAF4, extending from the degQ start codon to codon 63 of comA, were aligned using the PILEUP alignment program (GCG Wisconsin package). This alignment was used as an input sequence for the PLOTSIMILARITY program. A 25-nucleotide window of comparison was moved one position at a time along the sequence, and the average similarity within the window is plotted at the middle position of the window. The overall average similarity is plotted as a dotted line. A genetic map is also shown above the plot. The membrane and linker domains of ComP are shown as shaded and solid bars, respectively. The linker domain is defined as extending from the C-terminal end of the 8th putative membrane-spanning segment to the 1st residue of the ComP H box.
- Fig. 2.
Alignment of ComX amino acid sequences. The conserved W residue is marked in boldface and was used to align the sequences with the ZEGA program (http://molsoft.com/services/help/intro.htm ). The following symbols indicate similar residues: #, hydrophobic residues; ∼, polar residue; ^, small residue; %, aromatic residue. Nonconserved residues are indicated by dots. The N-terminal alanine inB. subtilis 168 ComX is underlined.
- Fig. 3.
Expression of srfA-lacZ by producer strains. β-Galactosidase activities were measured in a set of isogenicB. subtilis strains expressing heterologouscomQXP genes. Time is in hours before or after the time of transition from exponential to stationary phase. The specificities of the producer strains in the two panels were as follows. (A) ▴, 168 (BD2833); ▾, RO-H-1 (BD2913); ▿, RS-B-1 (BD2914); ⧫, B. natto NAF4 (BD2915). (B) ×, RO-FF-1 (BD2939); ▵, RO-B-2 (BD2936); ■, RO-C-2 (BD2937); □, RO-OO-2 (BD2947).
- Fig. 4.
Specificity of the QS response. Isogenic B. subtilis tester strains that do not produce ComX were grown in the presence of conditioned media prepared from isogenic producer strains. β-Galactosidase expressed from a srfA-lacZ reporter construct was assayed during growth. The following tester strains were used: BD2876 (derived from B. subtilis 168) (A), BD2877 (derived from B. natto NAF4) (B), BD2962 (derived fromB. mojavensis RO-H-1) (C), and BD2963 (derived from B. mojavensis RO-C-2) (D). Conditioned media were from strain 168 (BD2833) (▴), RO-H-1 (BD2913) (▾), RS-B-1 (BD2914) (▿), B. natto NAF4 (BD2915) (⧫), RO-B-2 (BD2936) (▵), and RO-C-2 (BD2937) (■). The open circles in panels C and D show the effect of adding fresh medium to the tester (panel D) or conditioned medium from the tester itself (panel C). Time is in hours before or after the time of transition from exponential to stationary phase. Note that the ordinate scales differ in the four panels.
- Fig. 5.
Competence pheromone produced in E. coli. TheB. subtilis 168 tester strain (BD2876) was grown in the presence of supernatant prepared from E. coli (ED318) carrying a plasmid with the B. subtilis 168 comQand comX genes. β-Galactosidase activities produced from asrfA-lacZ reporter construct were assayed during growth. BD2876 was grown in the presence of 10-fold (▵), 20-fold (▾), and 100-fold (□) dilutions of the E. coli supernatant. The activities of the B. subtilis producer (BD2833) (▴) and tester (○) strains grown in fresh medium are included for comparison. (The open circles are obscured by the solid squares). Conditioned medium from an isogenic E. coli strain (ED319) carrying thecomQ and comX genes in reversed (nonexpressing) orientation (■) exhibited no activity.
Tables
- Table 1.
Strains and plasmids used in this study
Strain or plasmid Genotype Source or reference B. subtilis OKB125 his leu met srfA::Tn917Ω OK120 (pTV55) Cmr 21 BD1658 his leu met comP::cat This work BD2833 his leu met srfA-lacZ (tet) This work BD2834 his leu met srfA-lacZ (tet)orf-2::kan This work BD2847 his leu met srfA-lacZ (tet) (comQ comX comPreplaced by genes from B. natto NAF4) BD2876 his leu met srfA-lacZ (tet)comQ::Km This work BD2877 his leu met srfA-lacZ (tet) (comQ::phl comX comP replaced by genes from B. natto NAF4) This work BD2911 his leu met srfA-lacZ (tet)amyE::xylR Pxyl-comK (ery) (ΔcomQXP)::Km This work BD2913 his met srfA-lacZ (tet)amyE::xylR Pxyl-comK (comQ comX comP replaced by genes from B. mojavensisRO-H-1) This work BD2914 his met srfA-lacZ(tet) amyE::xylR Pxyl-comK(comQ comX comP replaced by genes from B. subtilis RS-B-1) This work BD2915 his met srfA-lacZ (tet) amyE::xylR Pxyl-comK (comQ comX comP replaced by genes fromB. natto NAF4) This work BD2935 his leu met srfA-lacZ (tet) amyE::xylR Pxyl-comK (cat) (ΔcomQXP)::Km This work BD2936 his met srfA-lacZ (tet)amyE::xylR Pxyl-comK (cat) (comQ comX comP replaced by genes from B. mojavensis RO-H-1) This work BD2937 his met srfA-lacZ (tet) amyE::xylR Pxyl-comK (cat) (comQ comX comP replaced by genes from B. mojavensis RO-C-2) This work BD2939 his met srfA-lacZ (tet)amyE::xylR Pxyl-comK (cat) (comQ comX comP replaced by genes from B. subtilis RO-FF-1) This work BD2947 his met srfA-lacZ (tet) amyE::xylR Pxyl-comK (cat) (comQ comX comP replaced by genes from B. subtilis RO-O-2) This work BD2962 his met srfA-lacZ (tet)amyE::xylR Pxyl-comK (cat) (comQ::pED345 comX comP replaced by genes from B. mojavensis RO-H-1) This work BD2963 his met srfA-lacZ (tet)amyE::xylR Pxyl-comK (cat) (comQ::pED346 comX comP replaced by genes from B. mojavensis RO-C-2) This work E. coli ED318 KC8 (PCR script with comQ comX fromB. subtilis 168 in + orientation) This work ED319 KC8 (PCR script with comQ comX from B. subtilis 168 in − orientation) This work KC8 hsdR leuB600 trpC9830 pyrF::Tn5 hisB463 lacΔX74 strA galU galK Clontech Plasmids pED302 PCR script with kancassette between degQ and comA This work pED345 pUS19 with internal comQ fragment ofB. mojavensis RO-H-1 inEcoRI/HindIII sites This work pED346 pUS19 with internal comQ fragment ofB. mojavensis RO-C-2 inEcoRI/HindIII sites This work pUS19 pUC19 with spc cassette between theNdeI and NarI sites This work - Table 2.
Wild-type Bacillus strains
Species Strain Group Reference B. subtilis 168 168 B. subtilis RO-OO-2 168 27 B. subtilis RO-FF-1 168 27 B. subtilis RS-B-1 W23 27 B. mojavensis RO-B-2 27 B. mojavensis RO-H-1 27 B. mojavensis RO-C-2 27 B. natto NAF4 34 - Table 3.
Summary of QS specificities
Producer specificity Response of the indicated tester strain to conditioned medium from the producer straina 168 RO-C-2 RO-H-1 NAF4 B. subtilis168 ++ ++ − − B. mojavensisRO-C-2 +/−b ++ − − B. subtilis RO-OO-2 − − − − B. subtilisRS-B-1 − − ++ − B. mojavensisRO-B-2 − − + − B. mojavensisRO-H-1 − − ++ − B. nattoNAF4 − − − ++ B. subtilisRO-FF-1 − − − −
