Inactivation of lsr2 results in a hypermotile phenotype in Mycobacterium smegmatis

Abstract

Mycobacterial species are characterized by the presence of lipid-rich, hydrophobic cell envelopes. These cell envelopes contribute to properties such as roughness of colonies, aggregation of cells in liquid culture without detergent, and biofilm formation. We describe here a mutant strain of Mycobacterium smegmatis, called DL1215, which demonstrates marked deviations from the above-mentioned phenotypes. DL1215 arose spontaneously from a strain deficient for the stringent response (M. smegmatis Delta rel(Msm) strain) and is not a reversion to a wild-type phenotype. The nature of the spontaneous mutation was a single base-pair deletion in the lsr2 gene, leading to the formation of a truncated protein product. The DL1215 strain was complicated by having both inactivated rel(Msm) and lsr2 genes, and so a single lsr2 mutant was created to analyze the gene's function. The lsr2 gene was inactivated in the wild-type M. smegmatis mc(2)155 strain by allelic replacement to create strain DL2008. Strain DL2008 shows characteristics unique from those of both the wild-type and Delta rel(Msm) strains, some of which include a greatly enhanced ability to slide over agar surfaces (referred to here as "hypermotility"), greater resistance to phage infection and to the antibiotic kanamycin, and an inability to form biofilms. Complementation of the DL2008 mutant with a plasmid containing lsr2 (pLSR2) reverts the strain to the mc(2)155 phenotype. Although these phenotypic differences allude to changes in cell surface lipids, no difference is observed in glycopeptidolipids, polar lipids, apolar lipids, or mycolic acids of the cell wall.

FIG. 1.

The lsr2 gene is required for normal M. smegmatis colony morphology. (A) 7H11 agar plate with a single DL1215 colony grown for 2 weeks. The colony is flat and smooth compared to the parental Δrel_Msm colony (see Fig. 2 in reference 9). (B) Sequence alignment of Lsr2 from strains mc²155 (wild type) and DL1215 of M. smegmatis. The underlined sequence indicates residue changes from the wild-type Lsr2 protein, and asterisks show residues missing in the mutant protein.

FIG. 2.

Lsr2 is required for biofilm formation. Strains are as follows: lane 1, mc²155; lane 2, Δrel_Msm strain; lane 3, DL2008; lane 4, DL2008/pLSR2; and lane 5, DL2008/pNBV1. When lsr2 is functional, strains are very hydrophobic, as evidenced by their ability to stay in suspension when Tween 80 is present (A) but clump in the absence of Tween (B). Strains without lsr2 function remain suspended without Tween. Pellicle formation was allowed to occur in standing cultures without Tween (C). Pellicles are missing if lsr2 is inactivated. Biofilm formation occurred in polystyrene tissue culture wells if lsr2 was functional (D). Crystal violet (CV) staining is shown relative to that for the wild type (WT) (100%). Error bars indicate standard deviations for 12 separate wells per strain.

FIG. 3.

M. smegmatis strains grown on low-agarose plates. (A) The effect of the agarose concentration on colony morphology is shown. 7H9 plates with decreased concentrations of agarose (0.8% and 0.3% [wt/vol]) were inoculated with 2-μl aliquots of M. smegmatis cultures grown to an OD₆₀₀ of 1.0. After 7 days of growth on plates, the edges of colonies were compared. On 0.3% agarose plates, the DL2008 colony showed fragmentation with separate large patches of cells (A4). (B) DL2008 forms a layer of “slime” on M63 plus 0.3% agarose, as visualized with a dissection microscope. The light refractive edge of the slime is shown (arrowheads), with patches of cells floating in it (arrows). Phase-contrast light microscope images of mc²155 cells (C) and DL2008 cells (D) at the perimeter of colonies. Magnification, ×200.

FIG. 4.

M. smegmatis strains inactivated for lsr2 have a hypermotility phenotype. (A) Several snapshots are shown for a 40-h period of a DL1215 colony migrating on M63 plus 0.3% agarose. Two-microliter aliquots of cells grown to an OD₆₀₀ of 1.0 were spotted on a humidified plate and allowed to grow. Relative migration of DL1215 is shown on the same plate with wild-type (WT) mc²155 and Δrel_Msm strains. (B) Hypermotility of DL2008 cells is eliminated by complementing the strain with the lsr2 gene (DL2008/pLSR2). Motility of DL2008 is unaltered by the vector alone (pNBV1).

FIG. 5.

TEM analysis of PTA-stained M. smegmatis cells adhering to Formvar-coated grids. (A) Wild-type M. smegmatis mc²155 growing on M63 plus 0.3% agarose plates shows the characteristic PTA-staining dark halos around the cell (arrow). (B) In addition to darkly stained halos near the bacterial surface (closed arrow), DL1215 cells also have a diffuse, cloudy halo extending far from their surfaces (open arrow). (C) When DL1215 cells are close together, their diffuse extracellular halos can merge together to form a large area with a distinct boundary (arrow). The insets in panel C are magnified in panels D and E. The edge of this diffuse halo (D) and an interior region of this halo (E) both show discrete structures resembling negative-staining rods with swellings at many of the ends (“lollipops”). (F) The edge of a PTA-stained DL1215 (lower left corner) cell shows these negative-staining structures extruding from the cell surfaces. (G) Extrusion of these negative-staining “lollipop” structures occurs from the poles in many cells and is shown here for two parallel DL2008 cells on 0.3% agarose plates. Bars = 1 μm for panels A to C, 100 nm for panels D to F, and 0.5 μm for panel G.

FIG. 6.

Inactivation of lsr2 is associated with resistance to bacteriophage lysis. The general transducing phage phAE159 was used to infect different M. smegmatis strains, and then plaque numbers were scored. Inactivation of lsr2 leads to a >4-fold decrease in susceptibility to phage infection (A). The presence of functional Lsr2 results in numerous clear plaques in strain DL2008/pLSR2 (B), while absence of Lsr2 results in fewer and turbid plaques in strain DL2008/pNBV1 (C). WT, wild type.

FIG. 7.

Lsr2 is associated with susceptibility to kanamycin. Zones of inhibition (cm) were measured around paper discs soaked with increasing amounts of kanamycin sulfate (A). Error bars represent standard deviations for three discs per antibiotic concentration. (B) Limiting-dilution RT-PCR analysis shows that expression levels of aph, the gene responsible for Kan^r, are higher for the lsr2 mutant background (DL2008) than for the parental mc²155 strain. Lanes 1 and 2 had cDNA as templates, and lane 3 had DNA as a template. RT-PCR analysis of 16S rRNA was performed to ensure equivalent amounts of cDNA were used as a template for PCRs. DNase-treated samples produced no PCR products (data not shown).

FIG. 8.

Maintaining plasmid DNA without selective pressure is dependent upon functional Lsr2. The extrachromosomal plasmids pNBV1 (vector) and pLSR2 both contain hygromycin resistance markers. When cells are repeatedly grown to stationary phase in the absence of antibiotic, hygromycin resistance is lost slowly if lsr2 is present either on the chromosome (mc²155) or on the plasmid (pLSR2). However, without a functional copy of lsr2 (DL2008/pNBV1; open squares), hygromycin resistance is lost very quickly. WT, wild type.