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Journal of Bacteriology, June 2008, p. 4291-4300, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.00023-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis ^,

Kriti Arora,¹ Danelle C. Whiteford,¹ Dalia Lau-Bonilla,¹ Christine M. Davitt,² and John L. Dahl^1*

School of Molecular Biosciences, Washington State University, Pullman, Washington 99164,¹ Franceschi Microscopy and Imaging Center, College of Sciences, Washington State University, Pullman, Washington 99164²

Received 4 January 2008/ Accepted 2 April 2008

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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 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 mc²155 strain by allelic replacement to create strain DL2008. Strain DL2008 shows characteristics unique from those of both the wild-type and 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 mc²155 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.

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Mycobacterium smegmatis is a fast-growing, saprophytic mycobacterial species. Although considered nonpathogenic, M. smegmatis provides a popular model for studying virulence mechanisms of slow-growing, pathogenic relatives such as Mycobacterium tuberculosis (9, 16, 37, 41) and Mycobacterium leprae (35, 42). An important aspect of mycobacterial pathogenesis is the ability of the pathogen to establish latent infections in hosts lasting for several years. Persistent M. tuberculosis bacilli in the host manifest drastic changes in gene expression that set the cells apart from actively growing tubercle bacilli (36, 40). One bacterial regulatory network that coordinates nutrient deprivation with adaptive metabolism is the stringent response. In mycobacteria this global regulatory system is controlled by a single gene called rel, and deletion of this gene in M. tuberculosis results in a severe defect in both long-term in vitro and in vivo survival (10, 30). We recently reported that the rel gene of M. smegmatis (rel_Msm) is involved in the regulation of cellular and colony morphologies (9). As seen with M. tuberculosis, the stringent response is required for long-term survival of M. smegmatis in culture, since the rel_Msm mutant readily dies over a month-long period while in stationary phase.

Here we report the appearance of a mutant strain, called DL1215, that arose spontaneously from the parental M. smegmatis rel_Msm strain. We selected for DL1215 by subjecting M. smegmatis rel_Msm cells to prolonged nutrient stress. This mutant does not represent a reversion to a wild-type phenotype, which is possible in bacteria deficient for the stringent response if suppressor mutations arise in their RNA polymerases (12). DL1215 also does not represent a contaminant, since its identity as M. smegmatis was confirmed by 16S rRNA sequencing.

The most striking phenotype of DL1215 is its ability to spread over soft-agar surfaces much faster (a trait referred to here as "hypermotility") than either the wild-type M. smegmatis mc²155 strain or the parental M. smegmatis rel_Msm strain. To our knowledge, this is the first report of a mycobacterial species demonstrating such a high rate of surface spreading motility. The genus Mycobacterium had been generally considered nonmotile until Roberto Kolter's laboratory demonstrated the abilities of M. smegmatis and Mycobacterium avium to spread on solid surfaces (22). This ability of M. smegmatis to spread was shown to directly correlate with the presence of glycopeptidolipids (GPLs) in the cell wall. Strains deficient in biosynthesis, transport, or acetylation of GPLs were unable to spread, and they produced colonies with a rougher phenotype than the wild-type strain (22, 31, 32). However, we show here that this hypermotility is independent of the GPL content of M. smegmatis and likely involves other cellular systems. This hypermotility directly correlates with inactivation of the lsr2 gene.

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Bacterial strains, culture media, and growth conditions. A list of the bacterial strains and plasmids used in this study is shown in Table 1. Liquid cultures were grown in 7H9 (Difco) medium supplemented with 0.2% glycerol and 0.05% Tween 80 unless stated otherwise. M. smegmatis strains were transformed with plasmid DNA by electroporation, as previously described (41). Transformants were selected for on Middlebrook 7H11 (Difco) agar medium containing hygromycin (50 µg/ml) or kanamycin (25 µg/ml) where appropriate.

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TABLE 1. Bacterial strains and plasmids

Generation of strain DL2008. Plasmid pKA0505 was constructed by first PCR amplifying the mutant lsr2 gene from strain DL1215 using forward primer LSR2F (5'-GATCTGAGCGTTGTTGATAG-3') and reverse primer LSR2R (5'-GTACCTGCCGTCCACTCTAA-3') (10). The 652-bp PCR product contained 199 bp upstream of the start codon and 108 bp downstream of the stop codon and was cloned into the EcoRI site of pDrive. The mutant lsr2 allele was then excised with BamHI and NotI and cloned into the BamHI-NotI site of the multiple cloning site in p2NIL to create pKA0504. The Hyg^r-P_Ag85-lacZ-P_hsp60-sacB marker cassette from pGOAL19 was released by PacI digestion and cloned into the PacI site of pKA0504 to create pKA0505. M. smegmatis mc²155 was transformed with pKA0505, and single crossover events were selected as blue colonies on 7H11 agar plates with 50 µg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside and 50 µg/ml hygromycin. Colonies were grown in 7H9, and double recombinants were selected for on 7H11 agar with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside and 10% sucrose (wt/vol). White colonies were verified for Kan^s and Hyg^s, and lsr2 was sequenced to confirm that allelic replacement had occurred. One colony was saved and was named strain DL2008.

Colony morphology analyses and motility assays. For colony morphology analyses, M. smegmatis strains were grown on Middlebrook 7H11 (Difco) agar medium. Motility assays were performed as described previously (22). Briefly, cells were cultured in 7H9 medium to mid-logarithmic phase (optical density at 600 nm [OD₆₀₀], 0.8 to 1.0) before spotting 2-µl aliquots onto motility medium consisting of M63 salts liquid medium (Difco) supplemented with 0.5% Casamino Acids, 0.2% glycerol, 1 mM MgCl₂, and 10 µM FeCl₂, solidified with agarose. The agarose concentration was either 0.3% or 0.8% (wt/vol). The inoculated plates were incubated for 24 h at 37°C in plastic bags containing moistened paper towels to ensure bacteria grew under humidified conditions.

Cell clumping, pellicle formation, and biofilm assay. The ability of M. smegmatis strains to clump in culture was analyzed in both 7H9 and M63 liquid media. Cells were grown to mid-log phase in medium with or without 0.05% Tween 80 in a 37°C shaker incubator. Cultures then sat at room temperature for 1 h to allow cell aggregates to settle. Pellicle formation was monitored, as previously described (6), using standing cultures with 0.05% Tween 80 at 37°C for 48 h. Biofilm formation on the sides of the wells in polyvinylchloride and polystyrene microwell plates was monitored as previously described (6, 31, 32).

Time-lapse imaging of spreading motility. Time-lapse photography was performed with 2-µl aliquots of mid-log-phase cultures spotted onto M63 medium with 0.3% agarose. Plates were incubated at 37°C in a chamber with constant humidity, and images were taken every 10 min for 48 h with a color charge-coupled-device camera (model CV-S3200; Jai Corporation). Video output from the camera was digitized with a DVBridge (Dazzle Inc). Images were captured at 10-min intervals with a Macintosh computer (iMac) using QuicktimePro 5.0.

Antibiotic sensitivity assays. The wild-type mc²155 strain and the lsr2-mutant DL2008 strain were transformed with an integrative plasmid (pMV306) containing a single copy of the kanamycin resistance gene (aph). Paper discs, soaked in kanamycin sulfate at amounts ranging from 0 to 1.5 mg, were placed on 7H11 plates swabbed with bacteria (grown in 7H9 plus 0.05% Tween 80 to an OD₆₀₀ of 0.1) and then allowed to incubate at 37°C. Zones of inhibition were observed and their diameters measured after 48 h of incubation at 37°C.

RT-PCR analysis. Relative levels of aph expression in different M. smegmatis strains was performed by limiting-dilution reverse transcription (RT)-PCR analysis, as previously described (4). The forward primer for 16S rRNA is 5'-CCGCAAGGCTAAAACTCAAA-3', and the reverse primer for 16S rRNA is 5'-TAACAAGGTAGCCGTACCGG-3'. The forward primer for aph is 5'-GGGAAAGCCACGTTGTGT-3', and the reverse primer for aph is 5'-AGGTCTGCCTCGTGAAGAAG-3'.

Plaque assays. Infections of M. smegmatis strains were carried out with the mycobacteriophage phAE159, as described previously (2). Briefly, 10-ml bacterial cultures were grown to mid-log phase (OD₆₀₀ of 0.8), washed twice in MP buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 24 mM MgCl₂, 2 mM CaCl₂) supplemented with 5% glycerol, resuspended in a 1/10 volume of MP buffer without glycerol, and then mixed with phage at a ratio of 1 x 10⁷ bacteria:15 phage. Following incubation at 30°C for 2 h, the phage-bacterium mixtures were added to top agar (0.3% agar [wt/vol]) and poured onto 7H11 plates. The resulting plaques were counted and photographed after incubation at 30°C for 48 h.

Electron microscopy. For transmission electron microscopy (TEM) analysis of whole cells growing on M63 plus 0.3% agarose, cells were lifted off plates and stained as previously described (22). Briefly, Formvar carbon-coated nickel grids were pressed gently onto the surfaces of colonies and allowed to absorb material for 3 to 5 s before being removed and rinsed twice with distilled H₂O by floating grids sample side down on 100-µl droplets. Samples were then stained either with 1% uranyl acetate, 2% phosphotungstic acid (PTA) (pH 7.5), or 1% ammonium molybdate for 1 min before rinsing grids again with distilled H₂O. Samples were then viewed using a Jeol TEM 1200 EX electron microscope (Jeol USA, Inc., Peabody, MA). Images were acquired using Soft Imaging Systems (SIS) Analysis 3.5 Imaging software and a MegaView III camera (Lakewood, CO). For TEM analysis of cell envelopes, cells were grown in liquid 7H9 medium with 0.05% Tween 80 and then prepared and visualized as previously described (8).

Preparation of lollipop-shaped structures from culture supernatants. Cells were grown in M63 liquid with 0.05% Tween 80 to an OD₆₀₀ of 1.5 before being subjected to the Waring-blender action of a FastPrep FP120 device (Thermo Savant) set at 6.5 for 45 s without glass beads present. Cultures were then centrifuged twice at 10,000 x g for 10 min to remove whole cells and large debris. Culture supernatants were then centrifuged at 50,000 x g for 3 h. All centrifugations took place at 4°C. Pellets were resuspended in 1/100-volume M63 plus 0.05% Tween 80, and 2-µl aliquots were dried on Formvar-coated grids before staining with 2% PTA for 5 min, after which grids were washed by floating on water droplets and viewed with a TEM.

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Selection of the DL1215 mutant. During the original competitive starvation assays between M. smegmatis mc²155 and the rel_Msm strain, the two strains were mixed together in equal concentrations and subjected to three different stress conditions: resuspension in Tris-buffered saline, gradual growth into stationary phase in 7H9 plus 0.05% Tween 80, or gradual depletion of oxygen in stationary 7H9 plus 0.05% Tween 80 cultures (9). At various time points, the mixtures of cells were serially diluted on 7H11 agar with hygromycin (to select for mc²155 cells containing a Hyg^r cassette) or with kanamycin (to select for M. smegmatis rel_Msm cells containing a Kan^r cassette inside the rel_Msm gene). After three weeks of competitive starvation under all conditions, the kanamycin-resistant cells resulted in two different colony morphologies. One type was wrinkled, dry, and nippled and resembled the original M. smegmatis rel_Msm strain previously described (9). The second colony type was flat, shiny, and mucoid in appearance. This flat, shiny phenotype was never seen in the wild-type mc²155 cells starved for equal lengths of time. This spontaneously appearing, shiny mutant was named DL1215, and when grown for more than 3 weeks on 7H11 agar, DL1215 developed a perimeter with ruffled edges (Fig. 1A). Growth rates of M. smegmatis mc²155, the rel_Msm strain, and DL1215 in 7H9 plus 0.05% Tween 80 liquid medium were identical (data not shown). 16S rRNA sequencing confirmed that DL1215 is a strain of M. smegmatis (data not shown).

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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 relMsm 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.

Complementation of the DL1215 strain with the lsr2 gene. Chen et al. recently reported that a transposon insertion into the lsr2 gene of M. smegmatis mc²155 results in an M. smegmatis strain (MS8444 mutant) with smooth colonies similar in appearance to strain DL1215 (6). To test if the DL1215 mutant reported here contains a defect in its lsr2 gene, the strain was transformed with pLSR2 (an Escherichia coli-Mycobacterium shuttle vector containing a wild-type copy of lsr2 under its native promoter) (Table 1). The resulting strain, DL1215/pLSR2, formed rough-looking colonies resembling those of the parental M. smegmatis rel_Msm strain (data not shown). When DL1215 was transformed with the cloning vector alone (pNBV1), however, there was no alteration in colony morphology. Sequence analysis of lsr2 confirmed that strain DL1215 contains a mutation in this gene. Loss of a single adenine nucleotide at the 40th codon resulted in a frameshift mutation leading to a stop codon eight codons downstream (Fig. 1B). The DL1215 Lsr2 protein was <42% of the length of the protein from the M. smegmatis mc²155 and rel_Msm strains. We believe that even though DL1215 is capable of producing a truncated version of the Lsr2 protein, this strain acts as though it has a null mutation for lsr2, since it is so similar in colony morphology (Fig. 1A) and hydrophobicity (Fig. 2) to what was reported previously for the MS8444 mutant containing a transposon insertion in the lsr2 gene (6).

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FIG. 2. Lsr2 is required for biofilm formation. Strains are as follows: lane 1, mc2155; lane 2, relMsm 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.

Inactivation of lsr2 in mc²155 by allelic replacement. Because strain DL1215 has both inactivated rel_Msm and lsr2 genes, it is not clear which unique DL1215 phenotypes are due to just lsr2 inactivation and which might be due to the double mutation. Also, because DL1215 arose spontaneously from the rel_Msm strain, it is possible that DL1215 has more than one unmarked mutation. To address both of these points, we replaced the lsr2 allele in mc²155 with the mutant lsr2 allele from DL1215 to create strain DL2008. DL2008 has a functional rel_Msm gene, and yet it displayed the same smooth-colony phenotype as DL1215 (data not shown).

Analysis of cell surface hydrophobicities and of pellicle and biofilm formations. Since MS8444 and DL2008 both have dysfunctional lsr2 genes, we examined the hydrophobicity of DL2008, as had previously been done for MS8444. Usually a detergent like Tween 80 (0.05% vol/vol) is required for growth of mycobacteria as dispersed cells in liquid cultures (Fig. 2A, all tubes). Growth in liquid medium without Tween 80 resulted in cell aggregation that led to reduced turbidity in shaking cultures of mc²155 and M. smegmatis rel_Msm (compare tubes 1 and 2 in Fig. 2A and 2B). However, strains DL2008 and DL2008/pNBV1 remained completely dispersed in culture even without Tween 80 (Fig. 2B, tubes 3 and 5), while DL2008/pLSR2 resembled the wild-type and rel_Msm strains in regard to clumping (Fig. 2B, tube 4). Dispersion of the DL2008 and DL2008/pNBV1 cultures without Tween 80 was likely due to reduced surface hydrophobicities of these cells.

Chen et al. have also shown the requirement of Lsr2 in pellicle formation at the air-liquid interface of stationary 7H9 liquid cultures (6). The DL2008 mutant was similarly tested in standing cultures of both M63 plus 0.05% Tween 80 (Fig. 2C) and 7H9 plus 0.05% Tween 80 (data not shown). In both media, DL2008 and DL2008/pNBV1 were not able to form thick pellicles above the liquid surfaces (Fig. 2C, tubes 3 and 5), although strain mc²155, the rel_Msm strain, and strain DL2008/pLSR2 did form visible surface pellicles above the air-liquid interface (Fig. 2C, tubes 1, 2, and 4).

The lack of an ability to form pellicles correlates with a defect in biofilm formation (6). Therefore, the ability to form biofilms on polyvinylchloride and polystyrene plastic surfaces was also tested. Figure 2D shows that both DL2008 and DL2008/pNBV1 did not appreciably adhere to the sides of polystyrene tissue culture wells, and consequently, these wells did not stain with crystal violet. These strains also did not adhere to polyvinylchloride wells (data not shown). This is in contrast to the other M. smegmatis strains tested, which were capable of forming biofilms that stained with crystal violet. Therefore, our results confirm the previously reported role of Lsr2 in regulating these cell surface-associated phenotypes. The requirement of Lsr2 for biofilm formation was further confirmed by performing floating biofilm assays with plastic petri dishes, as described previously (25) (data not shown).

Cell motility on plates with low concentrations of agarose. A phenotype not reported for MS8444 is its surface sliding ability. Previously, Roberto Kolter's laboratory has shown that mc²155 is motile on media with 0.3% agarose and that this motility was dependent upon the ability of M. smegmatis to produce and acetylate GPLs (31, 32). Based upon the observation of the smooth-colony nature of the lsr2 mutant (Fig. 1) (6), we compared its motility to that of other isogenic strains (Fig. 3A). Two-microliter aliquots of mid-log-phase cultures (OD₆₀₀ of 1.0) were spotted on M63 plates with 0.8% or 0.3% (wt/vol) agarose. At 0.8% agarose, all colonies were intact and had a smooth circumference (Fig. 3A1, A3, and A5), while at 0.3% agarose, DL2008/pNBV1 lacked the defined colony edges seen for the other strains (Fig. 3A4). The edges of the M. smegmatis mc²155 and DL2008/pLSR2 colonies showed that cells were all adjacent to one another (Fig. 3A2 and 3A6, respectively). However, the DL2008/pNBV1 colony edge appeared completely fragmented, with groups of cells migrating as independent "rafts" apart from one another (Fig. 3A4). These "rafts" of DL2008/pNBV1 cells appeared to be floating in a layer of slime. When a light source was positioned at an angle from a DL2008 colony on M63 plus 0.3% agarose, a fragmenting pattern of cells was seen (Fig. 3B, arrows) in addition to the light source reflecting off the edge of a shiny river of slime extending beyond the edges of the colony (Fig. 3B, arrowheads). Figures 3C and D show colony edges of the mc²155 and DL2008 strains, respectively, on M63 plates with 0.3% agarose. Whereas mc²155 cells grew on top of each other and the colony edge was smooth (Fig. 3C), DL2008 cells existed in a monolayer, and aggregates of cells were seen dispersed from the main colony on the solid medium (Fig. 3D).

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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, x200.

Time-lapse photography of strain mc²155, the M. smegmatis rel_Msm strain, and strain DL1215 migrating on the same M63 plus 0.3% agarose plate is shown in Fig. 4A. Over a 40-h period, DL1215 showed motility at the rate of 31.25 µm/min, which was faster than mc²155 or rel_Msm growing on the same plate (see a QuickTime movie in the supplemental material). Strain DL2008 showed the same level of "hypermotility" as DL1215, and complementation of DL2008 with lsr2 resulted in a loss of this excessive motility (Fig. 4B). These results indicate that Lsr2 plays a role in regulating surface spreading in M. smegmatis. Interestingly, this effect of Lsr2 was influenced by the stage of growth. The excessive motility of DL1215 and DL2008 was exhibited by mid-log-phase cells, whereas stationary-phase cultures (3 days old) spotted onto M63 plus 0.3% agarose did not show any migration in excess of that of mc²155 (data not shown).

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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 relMsm 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).

Extracellular structures detected by TEM of whole cells. In order to better observe cells at the periphery of the motile mass, TEM analysis was performed. Whole cells were lifted from the surfaces of M63 plus 0.3% agarose plates near the edges of spreading colonies (Fig. 5). The mc²155 cells at the periphery of colonies had PTA-stained, extracellular material associated with them, as has been previously described (Fig. 5A) (22). This dark extracellular staining was also evident for numerous DL1215 cells lifted from plates (Fig. 5B). However, in addition to this darkly staining material, the DL1215 cells also had diffuse matrices surrounding them (Fig. 5B). These diffuse matrices from several different cells were capable of joining together to form a connective layer (Fig. 5C). When viewed under higher magnifications, these layers seemed to be comprised of negatively stained structures resembling bacteriophage heads connected to tails (Fig. 5D, E, and F). These "lollipop" structures were visible only in the DL1215 and DL2008 cells (Fig. 5G) and only when these cells were stained with PTA. Uranyl acetate and ammonium molybdate staining did not allow visualization of these structures (data not shown). These structures were absent from mc²155, rel_Msm, and DL1215/pLSR2 cells and from DL2008/pLSR2 cells (data not shown). Such extracellular structures were not reported by Chen et al., who first described an lsr2 mutation in M. smegmatis (6). Formvar-coated grids used to lift material from areas of the plate containing light-refractive slime but no visible cells (see Fig. 3B) did not have any PTA-staining material (data not shown). When mc²155, rel_Msm, DL2008/pNBV1, and DL2008/pLSR2 cells were grown in M63 liquid with 0.05% Tween 80, "lollipop" structures were observed only in the culture supernatant of DL2008/pNBV1 and not in that of any of the other three strains (data not shown).

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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.

Taken together, these findings indicate that Lsr2 has a role in regulating the production of these extracellular structures from M. smegmatis cells. It cannot be said with certainty if these structures are involved in the spreading of M. smegmatis on soft agar surfaces. However, the appearance of these structures in association with the hypermotile strains DL1215 and DL2008 suggests that they may represent an as yet unidentified mechanism of mycobacterial motility.

Loss of Lsr2 increases resistance to mycobacteriophages. Loss of hydrophobicity in DL2008 raises the possibility of an alteration in cell envelope permeability. Parish et al. have previously characterized an M. smegmatis mutant with increased hydrophobicity and altered cell envelope permeability. This mutant was initially characterized as producing unusually large plaques when infected with various mycobacteriophages (27). Based upon this reported link between cell hydrophobicity, cell permeability, and phage infection, the M. smegmatis strains in this study were tested for infection by the general transducing mycobacteriophage phAE159 (Fig. 6) and phAE86 (data not shown). The mc²155, rel_Msm, and DL2008/pLSR2 strains all produced comparable numbers of plaques (Fig. 6A). However, phAE159 infections of DL2008 and DL2008/pNBV1 produced lower numbers of plaques, which were also more turbid. Appearances of phAE159 plaques on lawns of DL2008/pLSR2 and DL2008/pNBV1 are shown in Fig. 6B and C, respectively. The reduced numbers of plaques for DL2008 and DL2008/pNBV1 could be due either to reduced entry/replication of phage in the host cells or to reduced attachment on the host cell surfaces. To test this hypothesis, TEM analysis was performed on whole cells allowed to adsorb phage. No difference was seen between the average numbers of phage visibly bound to cell surfaces for M. smegmatis strains with or without functional lsr2 (data not shown). Therefore, we believe the reduced numbers of plaques in DL2008 and DL2008/pNBV1 were due to a reduction in phage entering or replicating within the host cells, indicating that Lsr2 has a role in regulating susceptibility to mycobacteriophage.

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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.

Lsr2 affects sensitivity to kanamycin. DL2008 was tested for susceptibility to a variety of antibiotics. Strain mc²155, the rel_Msm strain, and strain DL2008 showed identical susceptibilities (diameters of zones of inhibition) to discs containing the following amounts of antibiotics: 10 µg penicillin, 1 µg oxacillin, 15 µg erythromycin, 30 µg cephalothicin, 30 µg vancomycin, 100 µg carbenicillin, 10 µg gentamicin, 30 µg amikacin, 30 µg amoxicillin-clavulanic acid, 5 µg ciprofloxacin, and 23.7 µg sulfamethoxasole-1.25 µg trimethoprim (data not shown). The three strains also showed no differences in susceptibilities to discs soaked with 1 to 1,500 ng of rifampin (data not shown).

One antibiotic susceptibility pattern not reported for strain MS8444 was for kanamycin sulfate. The original M. smegmatis rel_Msm strain had a kanamycin resistance cassette inserted into its rel_Msm gene (9). The spontaneous mutant, DL1215, that arose from this had greatly enhanced kanamycin resistance compared to the rel_Msm strain (data not shown). It was therefore hypothesized that Lsr2 played a role in resistance to this antibiotic. The wild-type strain mc²155 and the lsr2-mutant strain DL2008 were made kanamycin resistant by transforming cells with an integrative plasmid (pMV306) carrying the Kan^r marker. Although they contain the kanamycin resistance cassette (aph gene) on the same position on the chromosome (insertion at the attB site), DL2008::pMV306(K) showed much greater resistance than the M. smegmatis mc²155::pMV306(K) parental strain (Fig. 7A). DL2008::pMV306(K)/pLSR2 zones of kanamycin inhibition resembled those of mc²155::pMV306(K) (Fig. 7A). Since the DL2008::pMV306(K)/pNBV1 strain showed no difference in kanamycin resistance from DL2008::pMV306(K), the lrs2 gene clearly plays a role in altering kanamycin susceptibility of this strain. RT-PCR analysis shows that the level of aph expression is higher in DL2008::pMV306(K) than in the parental mc²155::pMV306(K) strain (Fig. 7C). This suggests Lsr2 exhibits some direct or indirect repression of aph expression.

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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 Kanr, 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).

Plasmid replication requires Lsr2. Without antibiotic selective pressure, episomes were lost quickly from the replicating M. smegmatis strain missing a functional lsr2 gene (Fig. 8). The pool of liquid-grown DL2008/pNBV1 cells lost its ability to grow on 7H10 agar plates with hygromycin (50 µg/ml) after only a few passages without selective pressure. However, M. smegmatis mc²155/pNBV1, mc²155/pLSR2, and DL2008/pLSR2 all showed only a modest loss of plasmid DNA after multiple passages in 7H9 liquid medium without hygromycin. These results suggest lsr2 may play some role in plasmid replication or plasmid segregation to daughter cells. We do not believe Lsr2 plays a significant role in chromosomal DNA replication since we observed DL2008 growing at the same rate as mc²155 cells in liquid culture (data not shown).

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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.

Analysis of cell envelope components. It has been reported that production of GPLs is needed for M. smegmatis motility (22, 31, 32). We analyzed GPL content for our lsr2 proficient and deficient strains. DL1215 is identical to its parental strain (rel_Msm) in GPL content (see the supplemental material). This is not surprising, because the previously reported lsr2-mutant strain, MS8444, also did not show any difference in its GPL profile (6) Therefore, our findings show that DL1215 hypermotility (Fig. 4A) is not due to an alteration of the GPL content. In addition to the GPL content, we discovered the mycolic acid compositions and polar lipid compositions of mc²155, the rel_Msm strain, and DL1215 to be identical (data not shown). This is also in agreement with results for the mutant MS8444 (6). However, MS8444 was shown to lack two apolar lipids identified as novel mycolate compounds (6). Two-dimensional thin-layer chromatography analysis of DL1215 apolar lipids in the current study failed to show any difference from the profiles of mc²155 and the rel_Msm strain (data not shown). Calcofluor white was used to stain surface-exposed carbohydrates of mc²155, the rel_Msm strain, and DL1215, but no differences were observed for these three strains (data not shown). As with the MS8444 mutant (6), TEM analyses of cell wall ultrastructures did not reveal any novel features in the DL1215 cell envelope (data not shown). Collectively, analysis of the cell envelope failed to reveal a structural or molecular explanation for the DL1215 colony morphology, cell hydrophobicity, or hypermotility.

Elevated levels of ribosomal protein L22 in lsr2-deficient strains. Although molecular analysis of cell envelopes revealed no differences between the rel_Msm and DL1215 strains for GPLs, mycolic acids, polar lipids, and apolar lipids, this was not true for whole-cell protein comparisons. After strain mc²155, the rel_Msm strain, and strain DL1215 were grown in liquid 7H9 with 0.05% Tween 80 to mid-log phase, their protein compositions were determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When equal amounts of protein from the lysates were compared, the protein profiles of the different strains were nearly identical with the exception of a single species (see the supplemental material). Elevated levels of a 17-kDa protein were present in DL1215 and DL1215/pNBV1 compared to results for mc²155, the rel_Msm strain, and DL1215/pLSR2. Peptide mass fingerprinting identified this protein as the L22 ribosomal protein. This result agrees with recently reported microarray analysis showing that the gene for L22 is up-regulated >2-fold in an lsr2-deficient M. smegmatis strain compared to results for a wild-type strain (7).

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This report describes a hypermotile strain of M. smegmatis that spontaneously arose from M. smegmatis rel_Msm cells exposed to conditions of oxygen and nutrient deprivation (9). This strain, M. smegmatis DL1215, is the result of a mutation rather than a phase variation. The nature of this mutation is a single base-pair deletion leading to a frameshift in the lsr2 gene (Fig. 1B). The lsr2 gene was subsequently inactivated in wild-type mc²155 to create strain DL2008. Lsr2 is a basic, cytosolic protein with no known functional motifs, but it has been shown to interact with DNA and serve as a regulatory protein (7). Although the biological role of Lsr2 has not been elucidated yet, it is recognized as an immunodominant T-cell antigen in M. leprae (20, 24). The transposon insertion inactivation of lsr2 from M. smegmatis mc²155 has previously been described for strain MS8444 (6). MS8444 and DL2008 have numerous similarities in their morphological phenotypes. However, here we report phenotypes not discussed before with respect to Lsr2, which include enhanced resistance to kanamycin, hypermotility, production of lollipop-shaped particles, and no change in the apolar lipid profile in DL2008.

The lsr2-deficient strain DL2008::pMV306(K) demonstrates approximately 2.5-fold greater resistance to 250 µg of kanamycin than mc²155::pMV306(K). This enhanced resistance could potentially be due to a number of different phenomena, including efflux of the antibiotic to lower the cytoplasmic concentration, elevated expression levels of the single aph gene in the cell, amplification of kanamycin targets, or decreased permeability of the cell envelope to the antibiotic. It is evident that Lsr2 has a role in regulating this effect, since complementation of DL2008::pMV306(K) with pLSR2 abolishes the ability to grow in high concentrations of the antibiotic (Fig. 7A). An alteration in cell permeability to kanamycin is not likely based upon a recent report by Colangeli et al. showing that M. smegmatis inactivated for lsr2 is not altered in cell permeability for hydrophobic or hydrophilic compounds (7). RT-PCR analysis indicates that enhanced kanamycin resistance is due to elevated aph expression in the absence of Lsr2 (Fig. 7B). Therefore, Lsr2 acts as either a direct or indirect repressor of aph expression.

Lsr2 has a distinct role in determining the cell envelope composition of M. smegmatis. As shown here and by Chen et al. (6), Lsr2 influences the ability of M. smegmatis to form biofilms, leads to the formation of smooth colonies, and alters the ability of M. smegmatis cells to clump in liquid culture. All of these are traits directly linked to hydrophobicity of mycobacterial cell walls. Biofilm and pellicle formation and aggregation of cells in culture have been shown to be altered by changes in cell surface hydrophobicities (6, 25). Therefore, it can be argued that Lsr2 inactivation leads to a reduction in the hydrophobicity of the DL1215 and DL2008 cell walls. In order to investigate this further, we performed biochemical analysis of cell envelope lipids of lsr2-deficient cells. It is not surprising that DL1215 shows no difference in its profiles for GPLs, polar lipids, and mycolic acids, since strain MS8444 also fails to show any differences in these classes of lipids (6). In contrast to MS8444, DL1215 does not show any difference in the profile of apolar lipids. This lack of difference in apolar lipids has been documented previously for another independent lsr2 mutant, as well (7).

The most interesting feature of the lsr2-inactivated strains is the enhanced motility ("hypermotility") on solid surfaces (Fig. 4). GPL-dependent motility has previously been demonstrated for M. smegmatis mc²155 (22, 31, 32). We observed a modest amount of motility for mc²155 in this study (Fig. 4A) (see the movie in the supplemental material). However, DL1215 exhibited up to a 12.5-fold greater motility rate (31.25 µm/min) than mc²155 (1.6 µm/min) and the smooth-colony mutant, Sm-1 (2.5 µm/min), described previously (22). Since the DL1215 mutant is not altered for GPL content (see the supplemental material), we believe this hypermotility involves cellular systems in addition to the GPLs. These cellular systems might be negatively regulated by Lsr2. It is possible that the lollipop-shaped ultrastructures observed here, while appearing in hypermotile strains, are not directly associated with the enhanced motility. Hypermotile strains might result from a reduced amount of adhesion to the agar surface, which leads to increased sliding.

Strain DL2008 produces smooth colonies characterized by increased mucoidy. Other mycobacterial species capable of generating smooth-colony morphotypes have been described (1, 5, 18, 19, 28). The smooth-colony phenotype of DL2008 is likely to be linked to changes in the hydrophobicity of the cell wall (Fig. 2). However, an interesting speculation is that colony morphology might be linked to production of lollipop-shaped extracellular structures resembling phage particles (Fig. 5). A precedent exists in M. smegmatis for lysogeny causing a shift from rough to smooth colony morphology (11, 14, 15, 17, 21, 34), and it has been proposed that M. smegmatis is a polylysogen (11, 14, 33). The correlation of these phage-like structures with inactivation of lsr2 suggests that an absence of Lsr2 may induce lysogens. The mycobacteriophage proteins Gp39 and Gp61 (from phage Cjw1 and Omega, respectively) share some homology to Lsr2, suggesting the gene could have arisen in mycobacteria by horizontal gene transfer (29). Annotation of the M. smegmatis genome has not revealed the presence of prophages. However, this absence of prophage identification could be due to the high degree of genetic diversity of mycobacteriophages and an inability to identify them by sequence gazing (29). The lollipop-shaped particles reported here have heads of various diameters and various tail lengths, as well as filamentous particles lacking any head structures. The correlation of these extracellular structures with inactivation of lsr2 suggests a possible link between their production and bacterial motility.

It has been reported that a gene encoding a type III restriction enzyme, MSMEG_1238, is 4.5-fold upregulated in the absence of Lsr2 (7). If this is true, then the MSMEG_1238 gene product can be expected to prevent foreign DNA from replicating in the bacterial cell. This may explain why DL2008 is more resistant to phage infection and subsequent plaque formation (Fig. 6). An alternative explanation for reduced plaque formation in DL2008 is that Lsr2 is a host cell protein needed for efficient phage replication.

 
We are indebted to Robert Kadner, Graham Haftull, David Dutton, Richard Friedman, and Patricia Hartzell for valuable comments made on this work. The plasmid pLSR2 and the bacteriophages phAE87 and phAE159 were generous gifts from Jun Lui and William Jacobs, Jr., respectively. We thank Derek Pouchnik of the WSU Sequencing Core Facility for assistance with DNA sequencing analysis. Eric Shelden assisted with time-lapse imaging of hypermotility.

This research was supported by grants from the American Lung Association (RG-022-N) and from the National Foundation for Infectious Diseases.

 
* Corresponding author. Mailing address: Washington State University, School of Molecular Biosciences, Abelson Hall, Room 301, Pullman, WA 99164. Phone: (509) 335-7719. Fax: (509) 335-1907. E-mail: johndahl{at}wsu.edu

Published ahead of print on 11 April 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, June 2008, p. 4291-4300, Vol. 190, No. 12
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