Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon

Abstract

The number and disposition of flagella harbored by eubacteria are regulated by a specific trait successfully maintained over generations. The genes governing the number of flagella in Bacillus subtilis have never been identified, although the ifm locus has long been recognized to influence the motility phenotype of this microorganism. The characterization of a spontaneous ifm mutant of B. subtilis, displaying diverse degrees of cell flagellation in both liquid and solid media, raised the question of how the ifm locus governs the number and assembly of functional flagella. The major finding of this investigation is the characterization of a newly identified dicistronic operon, named swrA, that controls both swimming motility and swarming differentiation in B. subtilis. Functional analysis of the swrA operon allowed swrAA (previously named swrA [D. B. Kearns, F. Chu, R. Rudner, and R. Losick, Mol. Microbiol. 52:357-369, 2004]) to be the first gene identified in B. subtilis that controls the number of flagella in liquid environments and the assembly of flagella in response to cell contact with solid surfaces. Evidence is given that the second gene of the operon, swrAB, is essential for enabling the surface-adhering cells to undergo swarming differentiation. Preliminary data point to a molecular interaction between the two gene products.

FIG. 1.

B. subtilis swrA operon. (A) Genetic organization of the swrA operon. (B) Amplification products obtained by RT-PCR on total RNA from strains PB1831 and PB5249, grown in solid (S) and liquid (L) media, by using primers yvzDF1-yvjDR2, designed to amplify an swrAA-swrAB overlapping region (lanes 1 to 4), and yvjBF1-yvzDR1, designed to amplify a yvjB-swrAA overlapping region (lanes 7 to 10). PCR amplifications of chromosomal DNA with primers yvzDF1-yvjDR2 (lane 5) and yvjBF1-yvzDR1 (lane 6). M, molecular size standard.

FIG. 2.

B. subtilis swrAB. (A) Nucleotide (lowercase letters) and deduced amino acid (uppercase letters) sequences of swrAB. The swrAB putative signal peptide sequence is in gray boxes, the putative transmembrane domains are in black boxes, and the PDZ domain is boldfaced and underlined. (B) ClustalW alignment of proteins similar to SwrAB from different bacilli. Asterisks indicate identical residues, colons indicate conserved substitutions, and dots indicate semiconserved substitutions. The conserved W necessary for SwrAB function is boldfaced and underlined. Swiss-Prot accession numbers, given at the left, refer to the following species: O34375_BACSU, B. subtilis; Q62PV4_BACLD, B. licheniformis ATCC 14580; Q812J4_BACCR, Bacillus cereus ATCC 14579; Q6HBA6_BACHK, Bacillus thuringiensis serovar Konkukian; Q81X32_BACAN, B. anthracis strain Ames Ancestor; and Q8ENJ4_OCEIH, Oceanobacillus iheyensis HTE831.

FIG. 3.

Morphological traits of the wild-type B. subtilis PB5249, the laboratory strain PB1831, and PB5249 derivatives carrying a deletion of either swrAA (PB5334) or swrA (PB5336). Stains PB5349 and PB5369 derive from PB5336 upon complementation (in the amyE locus) with a functional copy of swrAA (containing an 8-bp A-T stretch) and a functional copy of swrA (containing an 8-bp A-T swrAA sequence), respectively. Panels show colony morphology (left) and cell flagellation (center) after 6 h of growth on swarm plates; extracellular flagellin (right panel) from cells grown in solid (S) and liquid (L) media was detected by Western blotting. DswrAB, ΔswrAB. Bars, 5 μm.

FIG. 4.

Swimming motilities of different B. subtilis strains.

FIG. 5.

Results of immunoblotting of cell lysates from strains PB5249 and PB1831, grown on solid (S) and in liquid (L) media, with an anti-SwrAA antiserum. M, molecular size standard.

FIG. 6.

Expression of GST gene-swrA and GST gene-swrAA fusions in E. coli. (A) Diagram showing the swrA operon and the gene fusions. Constructs were as follows: 1, GST gene-swrAA fusion; 2 through 7, GST gene-swrA fusions with either native swrAB (2) or mutated swrAB carrying a deletion corresponding to a portion of the PDZ domain (3), the first transmembrane domain (TD1) plus the signal peptide (PepSig) (4), the first transmembrane domain only (5), or transmembrane domains 1 through 5 and the signal peptide (6) or harboring a point mutation producing a change from tryptophan (W) to arginine (R) (7). (B) SDS-PAGE of whole-cell homogenates from E. coli clones transformed with constructs 1 through 7. (C) SDS-PAGE of purified GST-SwrAA fusion proteins obtained with constructs 1 and 2. (D) Western blot of the same amounts of purified SwrAA incubated at 37°C for 1 or 2 h without and with plasma membranes isolated from strains PB5336 (ΔswrAA ΔswrAB), PB1831 (laboratory strain carrying a 9-bp A · T stretch of swrAA), and PB5249 (wild-type strain carrying an 8-bp A · T stretch in swrAB). M, molecular size standard.