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Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. Exponentially growing populations of Bacillus subtilis contain two morphologically and functionally distinct cell types: motile individuals and nonmotile multicellular chains.

Morphological heterogeneity is explained by the expression of LytF that is necessary and sufficient for cell separation. Moreover, LytC is required for motility but not at the level of cell separation or flagellum biosynthesis. Rather, LytC appears to be important for flagellar function, and motility was restored to a LytC mutant by mutation of either lonA , encoding the LonA protease, or a gene encoding a previously unannotated swarming motility inhibitor, SmiA.

Growing populations of Bacillus subtilis are heterogeneous in cell morphology Some members of these populations grow as single cells that are motile, whereas other cells in the populations grow in multicellular, nonmotile chains. The difference in motility between the two cell types is controlled at the level of gene transcription. Cells that are ON for flagellin expression complete flagellum assembly and constitute the motile subpopulation.

Cells that are OFF for flagellin expression do not complete flagellum biosynthesis and constitute the nonmotile subpopulation. The dimorphism of single cells and chaining cells in heterogeneous populations has not been resolved but is likely related to autolysin activity. Autolysins are bacterial enzymes that hydrolyze and remodel the peptidoglycan found in the bacterial cell wall 38 , During cell division, the cell wall grows inward by a process called septation to divide the mother into two identical daughters.

The daughter cells subsequently remain joined by a shared layer of peptidoglycan, and autolysins are required for daughter cell separation 1 , 10 , 11 , Autolysins have also been implicated in cell wall turnover, sporulation, and motility 12 , 33 , 34 , We determine that the autolysins are expressed in the same subpopulation that expresses the flagellar filament. In an undomesticated strain background B.

Finally, we demonstrate that the requirement of autolysins for motility is related to flagellar function rather than to flagellar biosynthesis or cell separation and that the motility defect could be genetically suppressed. Plates containing freshly prepared LB agar fortified with 0. The India ink marked the origin of a colony, and the swarm radius was measured relative to the origin. For consistency, an axis was drawn on the back of each plate, and the swarm radius was measured along this axis Fluorescence microscopy was performed with a Nikon 80i microscope with an Excite metal halide lamp.

Green fluorescent protein GFP and Alexa Fluor C 5 maleimide fluorescent signals were viewed using a phase-contrast objective Nikon Plan Apo X and were visualized using a C-FL HYQ fluorescein isothiocyanate filter cube excitation filter wavelength, to nm; barrier filter wavelength, to nm.

Images were captured with a Photometrics Coolsnap HQ2 camera in black and white, false colored, and superimposed using Metamorph image software. For fluorescent microscopy of flagella, 0. Three microliters of the suspension was placed on a microscope slide and immobilized with a poly- l -lysine-treated coverslip 4.

Images were captured with Metamorph software. All constructs were first introduced into domesticated strain PY79 by natural competence and then transferred to the background using SPP1-mediated generalized phage transduction All plasmids used in this study are listed in Table S1 in the supplemental material. All primers used in this study are listed in Table S2 in the supplemental material.

The two fragments were then simultaneously ligated into the EcoRI and BamHI sites of pMiniMAD, which carries a temperature-sensitive origin of replication and an erythromycin resistance cassette, to generate pKB The integrated plasmid was then transduced into strain DS Dilution and outgrowth were repeated two more times.

Individual colonies were patched on LB agar plates and LB agar plates containing mls to identify mls -sensitive colonies that had evicted the plasmid. The digested fragment was then cloned into the EcoRI and BamHI sites of pDG containing an erythromycin resistance cassette mls and a polylinker between the arms of the thrC gene The digested fragment was then cloned into the EcoRI and HindIII sites of pDG containing an erythromycin resistance cassette mls and a polylinker between the arms of the thrC gene The cyan fluorescent protein CFP gene, codon optimized for B.

The two fragments were then simultaneously ligated into the EcoRI and BamHI sites of plasmid pDG, which carries an erythromycin resistance marker and a polylinker between two arms of the thrC gene The culture was then serially diluted and spread on prewarmed LB agar plates fortified with 1.

The colonies were pooled and spotted in the center of a 0. Most of the cells remained nonmotile, but rare motile flares emerged from the central colony and were clonally isolated.

Cells from the motile flares likely contained transposons linked to the mutation of interest. To confirm that the transposon was linked to the suppressor mutation, a lysate was generated for the suppressor mutant and the transposon was transduced into the parent strain lacking the suppressor. Transposon insertion sites were identified by partially degenerate touchdown PCR using primer , hybrid degenerate primer , ng of purified chromosomal DNA, and Phusion polymerase New England Biolabs Observation of exponentially growing cells by fluorescence microscopy revealed that expression of lytF was heterogeneous in the population; it was ON in single cells and OFF in long chains Fig.

The same subpopulation of cells that expressed lytF also expressed flagellin Fig. The LytF autolysin and flagellin are expressed in the same subpopulation of cells. Arrowheads indicate the location of a chain. Flagellin expression in the majority of cells in an exponentially growing population of laboratory strains like strain PY79 is OFF because of a loss-of-function frameshift mutation in the swrA gene 5 , 16 , 18 , In contrast, the ancestral undomesticated strain B.

As observed for flagellin expression, a larger population of the undomesticated strain than of the laboratory strain expressed the P lytF promoter Fig. We now realize that laboratory strains grow primarily as chains because a majority of the cells in populations fail to express the autolysin genes, and this may obscure cell separation defects of autolysin mutants Fig.

We therefore investigated the roles of LytC, LytD, and LytF in chain separation in an undomesticated strain, the majority of cells of which express the three autolysins and grow as separate individuals Fig.

Cells in which lytC or lytD was mutated grew predominantly as individuals, but cells in which lytF was mutated grew predominantly as chains Fig. Double mutation of the lyt genes had additive phenotypic effects Fig. Simultaneous mutation of lytC , lytD , and lytF was required to produce a strain that grew constitutively as long chains and resembled a strain in which sigD was mutated Fig.

The lytF mutant has a severe defect in cell separation. Phase-contrast microscopy was performed with cells having the genotypes indicated grown to mid-log phase OD , 0. The bar graph shows a quantitative measure of chaining. More than cells were counted for each strain. In contrast, induction of lytF with 1 mM IPTG resulted in cell separation but also caused severe cell morphology defects and cell lysis Fig. Expression of lytF is sufficient to dissolve chains.

A Phase-contrast microscopy of cells grown to mid-log phase OD , 0. Undomesticated strains of B. In the undomesticated strain, mutation of lytC , but not mutation of lytD or lytF , resulted in a reduction in the rate of swarming motility Fig. Swarming motility was completely abolished in cells with double mutations in lytC and either lytD or lytF Fig.

In general, the autolysin mutants showed a comparable reduction in a swimming motility assay performed with 0. We conclude that LytC is required for efficient swarming motility. We note that the requirement for LytC for motility was not related to cell separation as the lytC lytD double mutant did not swarm despite growing predominantly as single cells.

Cells in which lytC is mutated have a swarming motility defect. Quantitative swarm expansion assays were performed with single A , double B , and triple C mutants with mutations in the lytC , lytD , and lytF autolysin genes. Each symbol indicates the average of measurements from three experiments.

Autolysins may be required for motility because they remodel the cell wall and this may permit flagellum assembly 8 , To monitor flagellum assembly, a version of flagellin that may be fluorescently labeled was inserted at the ectopic amyE locus in various genetic backgrounds [ amyE :: P hag - hag TC ] 4.

Flagellar staining and fluorescence microscopy revealed assembly of copious flagella in the wild type Fig. All single autolysin mutants and all combinations of double mutants produced flagella at a density comparable to that produced by the wild type data not shown , and the lytC lytD lytF triple mutant grew in chains but was nonetheless proficient for flagellum biosynthesis Fig.

In contrast, the chains of either sigD or swrA null mutants failed to produce flagella because, in these cases, the flagellin gene was not expressed Fig. Consistent with previous reports, the single cells in a swrA mutant population synthesized flagella, and there appeared to be fewer flagella per cell in this population than in a wild-type population 5 Fig. Cells with simultaneous lytC , lytD , and lytF mutations are proficient for flagellar assembly. The images are fluorescence microscopy images of cells grown to mid-log phase OD , 0.

Each strain expresses the modified flagellar filament protein Hag TC. The motility defect of a lytC lytD double mutant could not be explained by either a failure to synthesize flagella or a failure to separate cells from a chain. Therefore, we took an unbiased genetic approach to identify suppressor mutations that could restore motility to the lytC lytD double mutant. The lytC lytD double mutant was mutagenized with transposons, and seven strains in which swarming motility was rescued were independently isolated.

Each transposon insertion was backcrossed into the lytC lytD parental background by using SPP1-mediated generalized transduction, and in each case the rescue of the motility phenotype was found to be inseparable from the insertion.

Four isolates had disruptions in the lonA gene encoding the LonA protease 37 , 41 Fig. The lonA gene is potentially cotranscribed and translationally coupled with the ysxC gene immediately downstream as the start codon of ysxC overlaps the stop codon of lonA The ysxC gene is reportedly essential in B.

To determine whether the transposon insertions in lonA were directly responsible for rescuing motility in the lytC lytD double mutant or whether the transposons were polar for ysxC expression, an in-frame markerless deletion of lonA was generated to minimize polar effects. Like the transposon insertion in lonA , the lonA deletion restored motility to the lytC lytD double mutant Fig.

Next, a complementation construct that ectopically restored lonA expression was generated. When the lonA complementation construct was introduced into the lytC lytD lonA triple mutant, motility inhibition was restored Fig. We conclude that the absence of lonA rescues motility in the lytC lytD double mutant and that this rescue is not due to polar effects on ysxC.

Mutations in lonA and smiA restore motility to a lytC lytD double mutant. A lonA genetic region and lonA complementation construct. The large open arrows indicate open reading frames. The bent arrows indicate promoters. The solid triangles indicate sites of transposon insertions that restored motility to the lytC lytD double mutant.

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