Regulating pilin expression reveals a threshold for S motility in Myxococcus xanthus

Abstract

An isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter was constructed in Myxococcus xanthus. The single-copy pilA gene encodes pilin, the monomer unit of M. xanthus type IV pili. To vary the level of pilA expression, we cloned its promoter in front of the lac operator, and a plasmid containing the construct was inserted into the chromosome of a DeltapilA strain. Induction of pilin expression increased smoothly as the dose of IPTG added to the culture was increased. IPTG-induced pilin rescued S motility of the DeltapilA strain to wild-type levels. The rate of S-motile swarming was found to be proportional to the number of pili (shear-sensitive pilin) produced rather than to the level of total pilin. In fact, S motility was not rescued until the total level of pilin was more than 50% of the wild-type level. This observation implies that a threshold concentration of pilin must be exceeded before the shear-sensitive material (pili) is polymerized in M. xanthus.

FIG. 1.

Assembly of an IPTG-inducible promoter. (A) Schematic drawing showing the pilA promoter from −855 to 1 bp inserted in front of a leader sequence containing the lac operator for binding of and repression by the lac repressor LacI. The KpnI and EcoRI restriction sites were used to insert a gene of interest (often pilA). (B) Base sequence of the leader and the lac operator. The sequence is preceded by the −24 and −12 boxes of the σ⁵⁴-dependent pilA promoter and the transcriptional start site at position 1. The locations of the lac operator and the RBS are indicated. The translational start site ATG is indicated by boldface type. (C) Insertion of the inducible promoter into the M. xanthus chromosome by homologous recombination within the pilA locus of DK10410. After electroporation of the plasmid into M. xanthus cells, a single homologous crossover produced a tandem duplication of the pilA promoter in which the upstream chromosomal region (thick line) drove the expression of the inducible promoter, whereas the 855-bp upstream region cloned in the vector (thin line) drove the expression of the native pilA gene, most often a ΔpilA allele.

FIG. 2.

Expression of P_{pilA-lacO-RBS}-pilA and rescue of S motility. (A) Immunoblot showing expression of pilin from P_{pilA-lacO-RBS}-pilA. Total cell lysates were prepared from 2.5 × 10⁸ cells spotted on 0.5× CTT agar plates, and aliquots were harvested after 8 h of incubation at 32°C. One microgram of total protein isolated from DK1622 (wild type), DK10410 (ΔpilA), DK12600 (DK10410::P_{pilA-lacO-RBS}-pilA), DK10404 (pilR-Ω3163), or DK12612 (DK10404::P_{pilA-lacO-RBS}-pilA) was loaded in each lane and reacted with anti-PilA antibody. The results are representative of the results of two independent experiments. (B) S motility assay. The abilities of strains DK1622 (wild type), DK10410 (ΔpilA), DK12600 (DK10410::P_{pilA-lacO-RBS}-pilA), DK10404 (pilR-Ω3163), and DK12612 (DK10404::P_{pilA-lacO-RBS}-pilA) to swarm by using S motility were investigated. A total of 2.5 × 10⁷ cells were spotted on 0.5× CTT-0.4% agar plates and incubated at 32°C. The plates were inspected and photographed after 48 h. The results are representative of the results of two independent experiments.

FIG. 3.

Induction of pilin in strain DK12601 grown vegetatively. (A) Immunoblot showing the time course of IPTG-induced expression of pilin from P_{pilA-lacO-RBS}-pilA. Total cell lysates were prepared from 2.5 × 10⁸ cells spotted on 0.5× CTT agar plates with or without 1 mM IPTG, as indicated at the bottom. Aliquots were harvested after incubation at 32°C for 0.5 to 8 h. One microgram of total protein isolated from DK1622 (wild type), DK10410 (ΔpilA), DK12600 (DK10410::P_{pilA-lacO-RBS}-pilA), or DK12601 (DK10410::P_{pilA-lacO-RBS}-pilA, lacI) was loaded in each lane and reacted with anti-PilA antibody. The results are representative of the results of three independent experiments. (B) IPTG induction of P_{pilA-lacO-RBS}-lacZ during growth. Specific activities of β-galactosidase were determined with 2.5 × 10⁸ cells of DK12603 (DK1622::P_{pilA-lacO-RBS}-lacZ, lacI) spotted on 0.5× CTT agar plates with 1 mM IPTG and incubated at 32°C for different times. (C) Immunoblot showing the induction of pilin expression from P_{pilA-lacO-RBS}-pilA as a function of IPTG concentration. Total cell lysates were prepared from 2.5 × 10⁸ cells spotted on 0.5× CTT agar plates with different amounts of IPTG. Aliquots were harvested after incubation at 32°C for 12 h. Then 0.5 μg of total protein isolated from DK1622 (wild type), DK10410 (ΔpilA), DK10415 (ΔpilS), DK12600 (DK10410::P_{pilA-lacO-RBS}-pilA), or DK12601 (DK10410::P_{pilA-lacO-RBS}-pilA lacI) was loaded in each lane and reacted with anti-PilA antibody. The results are representative of the results of three independent experiments. (D) Relative expression of P_{pilA-lacO-RBS}-pilA and P_{pilA-lacO-RBS}-lacZ. Specific activities of β-galactosidase were determined with cells of DK12603 (DK1622::P_{pilA-lacO-RBS}-lacZ lacI) spotted on 0.5× CTT agar plates with different amounts of IPTG and incubated for 12 h at 32°C. The values are expressed relative to the expression of DK12602 (DK1622::P_{pilA-lacO-RBS}-lacZ) (defined as 100%) and are plotted as a function of the IPTG concentration. Each value is the average of the values from two independent experiments, and the error bars indicate standard deviations. The expression of pilin from P_{pilA-lacO-RBS}-pilA in DK12601 in panel C was quantitated by measuring band intensities, and the values are expressed relative to pilin expression in DK1622 and plotted as a function of the IPTG concentration. Each value is the average of the values from three independent experiments, and the error bars indicate standard deviations. Symbols: ▵, β-galactosidase (P_{pilA-lacO-RBS}-lacZ); ▪, pilin (P_{pilA-lacO-RBS}-pilA). wt, wild type.

FIG. 4.

IPTG induction of lacZ from P_{pilA-lacO-RBS}-lacZ during development: specific activity of β-galactosidase expressed in DK12602 (P_{pilA-lacO-RBS}-lacZ) and DK12604 (P_{pilA-lacO-RBS}-lacZ, P_pilA-lacI) during development. Specific activities of β-galactosidase were determined in cells after exposure to starvation in MC7 buffer with or without 1 mM IPTG for different times. Symbols: ▴, DK12602; □, DK12604 with IPTG; ⋄, DK12604 without IPTG. The specific activities of β-galactosidase are expressed in nanomoles of o-nitrophenol produced from o-nitrophenol-β-d-galactoside per minute per milligram of protein. Each value is the average of the values from two independent experiments, and the error bars indicate standard deviations.

FIG. 5.

Dose-dependent induction of S motility in DK12601. Strain DK12601 is DK10410::P_{pilA-lacO-RBS}-pilA lacI. Strains DK1622 (wild type), DK10410 (ΔpilA), and DK12600 (DK10410::P_{pilA-lacO-RBS}-pilA) were included as positive and negative controls. A total of 2.5 × 10⁷ cells were spotted on 0.5× CTT-0.4% agar plates and incubated at 32°C. The concentration of IPTG used (millimolar) is indicated in the top right corner of each panel. The swarming was inspected after 48 h. The results are representative of the results of three independent experiments.

FIG. 6.

Relationship between the amount of pilin synthesized and S motility. The levels of S motility at different IPTG concentrations, as shown in Fig. 5, are plotted as a function of the levels of pilin at the corresponding IPTG concentrations shown in Fig. 3C and D. The values are expressed relative to wild-type S motility and wild-type pilin production. Each value is the average of the values from three independent experiments, and the error bars indicate standard deviations. wt, wild type.

FIG. 7.

Cell-cell agglutination assay. Cells of DK1622 (wild type), DK10410 (ΔpilA), and DK12601 (DK10410::P_{pilA-lacO-RBS}-pilA lacI) were grown at 32°C overnight in CTT medium with or without 1 mM IPTG, and the OD₅₅₀ was adjusted to 0.6 with CTT broth. The absorbance after 120 min was determined relative to the initial absorbance for each strain. Solid bar, DK1622; open bar, DK10410; shaded bars, DK12601. Each value is the average of the values from three independent experiments, and the error bars indicate standard deviations.

FIG. 8.

Level of piliation in DK1622 (wild type) and DK12601 (DK10410::P_{pilA-lacO-RBS}-pilA lacI) measured by sensitivity to shearing. Material removed from cells by shearing was purified from DK1622 and DK12601 cultures after incubation at 32°C on 0.5×CTT agar plates for 12 h. Material purified from 100 μg of cells was loaded in each lane and reacted with anti-PilA antibody. The results are representative of the results of three independent experiments.