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Research Article

Deletion of the histone-like protein (Hlp) from Mycobacterium smegmatis results in increased sensitivity to UV exposure, freezing and isoniazid

Danelle C. Whiteford, Jesse J. Klingelhoets, Michael H. Bambenek, John L. Dahl

  • 1Sigma-Aldrich, Biotech Process Development, St Louis, MO 63103, USA
  • 2Department of Biology, University of Minnesota Duluth, Duluth, MN 55812, USA
  • Correspondence
    John L. Dahl
    jldahl{at}d.umn.edu

    • Microbiology 2011; 157(2):327–335 · https://doi.org/10.1099/mic.0.045518-0

    View at publisher PubMed

    Abstract

    Adaptation to environmental stress is an important survival characteristic of any bacterial species. As a soil-dwelling saprophyte, Mycobacterium smegmatis is exposed to factors such as UV light and rounds of freezing and thawing that occur in temperate climates. Numerous studies in Escherichia coli have linked histone-like proteins to stress resistance and adaptation. We hypothesized that the ‘histone-like’ protein Hlp might likewise be involved in the stress response of M. smegmatis. The hlp gene was inactivated and the M. smegmatis Δhlp strain was found to be more susceptible to UV light and to the stress created by repeated cycles of freezing and thawing. In addition, loss of Hlp altered the colony morphology and allowed the organism to grow dispersed in the absence of a detergent, suggesting changes in the cell wall composition. As cell wall changes could affect permeability to certain antibiotics, the susceptibility of M. smegmatis Δhlp to kanamycin, rifamipicin, ethambutol and isoniazid (INH) was tested. M. smegmatis Δhlp was more susceptible to INH, but loss of Hlp did not affect susceptibility to the other antibiotics tested. This suggests that the increased sensitivity of M. smegmatis Δhlp to INH was unlikely to be the result of alterations in cell permeability.

    Edited by: G. R. Stewart

    INTRODUCTION

    Mycobacterium smegmatis is a non-pathogenic, fast-growing mycobacterial species that is a popular model for studying slow-growing mycobacterial pathogens. It is a soil-dwelling species that has also been isolated from water and vegetation found in sphagnum moss bogs (Kazda, 2000). In these environments, M. smegmatis is naturally exposed to environmental stresses that without adaptive survival mechanisms would be lethal. The lipid-rich cell envelope of Mycobacterium provides some protection against stress by acting as a barrier to the environment (Brennan & Nikaido, 1995). However, specific stress responses allow the cell to sense environmental insults and alter gene regulation to produce cellular and physiological changes that increase resistance. In bacterial species, a class of ‘histone-like’ proteins has been linked to the survival of numerous environmental stresses, such as UV light (Li & Waters, 1998; Miyabe et al., 2000), cold shock (Wada et al., 1988), heat shock (Wada et al., 1988) and lack of nutrients (Claret & Rouviere-Yaniv, 1997).

    Bacterial histone-like proteins are small, highly basic proteins that are typically found at high abundance in cells (Thanbichler et al., 2005). These proteins display similarities to eukaryotic histones in their ability to bind DNA and influence gene regulation (Arfin et al., 2000; Parekh & Hatfield, 1996; Swinger & Rice, 2004). M. smegmatis possesses a histone-like protein called Hlp (also known as Hup, DNA-binding protein HU, CipMa and MDP1) (Katsube et al., 2007; Lee et al., 1998) that is encoded by locus MSMEG_2389. This protein contains an N-terminal domain with sequence homology to the HU proteins of Escherichia coli (Mukherjee et al., 2008), the best-studied family of histone-like proteins in bacteria. Hlp differs from other bacterial histone-like proteins in that it contains a C-terminal domain composed of prolines, alanines and lysines, resembling the eukaryotic histone H1 family of proteins (Mukherjee et al., 2008). Both the C-terminal and N-terminal domains of Hlp are involved in binding DNA with high affinity (Mukherjee et al., 2008). Homologues of the M. smegmatis hlp have been found in most mycobacterial species, including pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae (Shires & Steyn, 2001).

    Hlp has been identified as the major protein upregulated during M. smegmatis dormancy (Lee et al., 1998). It has also been identified as the major cold-shock-inducible protein in M. smegmatis (Shires & Steyn, 2001). Its homologue in M. tuberculosis, HupB, has been found to be upregulated by iron limitation (Yeruva et al., 2006) and by the stringent response (Dahl et al., 2003). Although HupB in M. tuberculosis is predicted to be an essential gene (Sassetti et al., 2003), Lee et al. (1998) were able to generate an hlp knockout in M. smegmatis. This deletion was found to have no effect on the viability of cultures grown for 2 weeks (Lee et al., 1998), but it did affect the ability of M. smegmatis to resume growth after being subjected to a 27 °C temperature reduction (Shires & Steyn, 2001).

    Induction of hlp homologues in response to stress and the apparent importance of Hlp in cold-shock adaptation suggest that the protein has a role in stress resistance. Because it can bind DNA and repress transcription in vitro (Matsumoto et al., 2000), it has been suggested that Hlp could be involved in regulating gene expression during a stress response (Mukherjee et al., 2008). In the current study we examined the effect of hlp deletion on the long-term survival of M. smegmatis in liquid culture and on the survival of cells exposed to environmental and chemical stresses.

    METHODS

    Bacterial strains, culture media and growth conditions.

    All M. smegmatis liquid cultures were grown in 7H9 (Difco) medium supplemented with 0.05 % Tween 80 and 0.2 % (v/v) glycerol, unless otherwise stated. For the long-term viability assay, cultures were gradually starved in 1.3 ml aliquots of 7H9+Tween 80 in Sarstedt tubes with gas-impermeable O-rings and little head space, and maintained in a 37 °C stationary incubator. At various time points, cells were gently vortexed with 0.1 mm diameter, sterile glass beads and serially diluted on 7H11 agar plates to determine c.f.u. ml−1.

    Deletion of hlp from M. smegmatis mc2155.

    Deletion of hlp from M. smegmatis mc2155 was performed by allelic replacement. Briefly, an approximately 700 bp PCR product (forward primer HusmF1, 5′-agtccacttaagcggcttccgggtcgtgatct-3′; reverse primer HusmR1, 5′-agtccatctagacgatggtgtcgacgacgttctcc-3′), corresponding to a chromosomal region directly upstream of hlp, was cloned into the AflII and XbaI restriction sites on pYUB854 (Jacobs et al., 1987) on one side of a hygromycin resistance (HygR) cassette. Another approximately 700 bp PCR product (HusmF2, 5′-agtccaccatggagaaggccgccgccaagaag-3′; HusmR2, 5′-agtccaagatctgaccggtcgacaccaagacggta-3′), corresponding to a region directly downstream of hlp, was inserted into the SpeI and HindIII restriction sites on the other side of the HygR cassette. The resulting plasmid was named pDW150. A marker cassette containing PAg85-lacZ-Phsp60-sacB was removed from pGoal17 (Parish & Stoker, 2000) and inserted into the PacI site of pDW150. This plasmid, named pDW151, was then treated with 100 mJ UV light cm−2 and electroporated into M. smegmatis mc2155.

    Following plating on 7H11 agar containing 40 μg X-Gal ml−1 and 50 μg hygromycin ml−1, transformants were selected that represented a single-crossover event (blue HygR colonies). The single-crossover transformants were then patched onto 7H11 plates with 50 μg hygromycin ml−1 and 10 % sucrose to screen for loss of sucrose sensitivity and of the lacZ marker. The resulting HygRSucIlacZ− colonies were considered double recombinants and verified for loss of hlp by PCR and Southern blot analyses.

    Complementation of hlp was performed using the integrative plasmid pMV306 (Stover et al., 1991). An 833 bp PCR product (HusmCF, 5′-cggggtaccccgaattcgccgcccacct-3′; HusmCR, 5′-cgcggatcccggcgtgccttttgcggaat-3′; KpnI restriction sites indicated by bold type) was generated to include both the entire hlp gene and approximately 150 bp upstream of the predicted translation start site. The PCR conditions were 94 °C for 3 min, and then 30 cycles of 94 °C, 30 s; 54 °C, 1 min; and 72 °C, 1 min. A final extension step of 72 °C for 7 min was included. This PCR product was then inserted into the KpnI and BamHI sites on pMV306 to create the construct for complementation called pMV306/hlp. This construct was electroporated into M. smegmatis Δhlp, where it integrated onto the chromosome to generate M. smegmatis Δhlp : : pMV306/hlp.

    Confirmation of hlp deletion from M. smegmatis.

    To confirm the loss of hlp, Southern blot analysis was performed as previously described (Sambrook et al., 1989). Genomic DNA from both M. smegmatis mc2155 : : pMV306 and Δhlp : : pMV306 was digested with NotI and ScaI for 24 h before separation by gel electrophoresis and transfer to a nylon membrane. A 306 bp PCR product (HusmPrF, 5′-cgacatcttccgggggaacg-3′; HusmPrR, 5′-ccggatcaggccgggagagt-3′) was amplified from the region directly upstream of the hlp gene on the M. smegmatis chromosome. This product was then used as a template for a PCR using a Digoxigenin (DIG) labelling kit (Roche). This reaction incorporated DIG-labelled dUTP into the PCR product, generating a probe for Southern blotting. This probe was allowed to hybridize membrane-bound DNA, and detection of the probe was performed using a Nitro-Blue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (NBT/BCIP) chromogenic reaction, as previously described (Wei et al., 2000).

    In addition to Southern blot analysis, deletion of hlp from the M. smegmatis chromosome was confirmed by PCR using primers that amplified a 208 bp internal region of hlp (HlpsmIntF, 5′-cgggttcggtgtcttcgagca-3′; HlpsmIntR, 5′-ggccttcttggcgggaccag-3′). As M. smegmatis Δhlp was generated using an allelic replacement strategy, this PCR was used to confirm the absence of the internal region of hlp from the chromosome. Coomassie-stained SDS-PAGE of late-exponential phase cultures was used to confirm the loss of the Hlp protein from M. smegmatis Δhlp.

    Transmission electron microscopy (TEM).

    TEM analysis was performed as previously described (Arora et al., 2008). Two-week-old colonies were swabbed off 7H11 agar and suspended in PBS. Cell suspensions were spotted onto Formvar carbon-coated nickel grids, which were stained with 1 % uranyl acetate before viewing with a JEOL TEM 1200 EX electron microscope.

    UV exposure.

    Aliquots (1 ml) of M. smegmatis cells grown to mid-exponential phase (OD600 0.6) were placed into sterile Petri dishes and exposed to increasing amounts of UV (5.0–15.0 J m−2) using a Bio-Rad GeneLinker UV box. Serial dilutions of treated cells were immediately performed and plated onto 7H11 agar. Results were reported as percentage survival compared with samples untreated with UV light.

    Isoniazid (INH) sensitivity assays.

    Sensitivity to INH (Sigma) was examined using both the Kirby–Bauer technique and determination of MIC. First, disks soaked with concentrations of INH from 0–20 μg ml−1 were placed on a lawn of cells swabbed from early exponential-phase cultures (OD600 0.2). After 48 h of incubation, diameters of the zones of inhibition were measured. The MIC was determined as described by Wallace et al. (1986). Briefly, cultures were grown to OD600 0.2 before diluting 1000-fold and placing 50 μl aliquots in triplicate into 96-well round-bottomed plates. Decreasing concentrations of INH (80–0.156 μg ml−1) were then added to the wells in 50 μl aliquots. Following a 48 h incubation period, wells were examined using a dissecting microscope for growth or no growth. Sensitivities to rifampicin, ethambutol and kanamycin were also tested using the two methods described above.

    Freeze–thaw assay.

    M. smegmatis cultures were grown to OD600 0.6 in 7H9+Tween 80. Aliquots (200 μl) were transferred to 1.8 ml cryovial tubes and frozen at −80 °C before removal and immediate thawing in a 37 °C water bath. This pattern was repeated 20 times before cells were serially diluted and plated onto 7H11 agar. c.f.u. were enumerated after 4 days growth and the percentage survival was calculated.

    Incubation of M. smegmatis in a sphagnum moss bog.

    Cryovial tubes containing 1.3 ml aliquots of M. smegmatis strains in 7H9+Tween 80 were buried 20 cm deep in the brown layer of a local sphagnum moss bog (N 4 °58.557′, W 09 °11.804′) for several months in 2009–2010. On the 15th of each month, three vials of each strain were extracted from the bog and viable bacteria determined by enumerating c.f.u. ml−1 on 7H11 agar plates. This bog is located 5 miles from Duluth International Airport and daily surface temperatures are recorded there for submission to the National Oceanic and Atmospheric Administration (NOAA).

    Acid-stress assay.

    Sensitivity of the M. smegmatis strains to low pH was determined by a method similar to that described by O'Brien et al. (1996). Briefly, cells were grown in Sauton's medium with 0.05 % Tween 80 adjusted to a pH of 6.8. At early exponential phase (OD600 0.015), cells were resuspended in 1 ml aliquots of either fresh Sauton's (pH 6.8) or Sauton's medium with a pH adjusted to a value of 5.0 or 4.0. Serial dilutions were performed on the pH 6.8 sample to determine c.f.u. ml−1 at the initial time point (0 h), which was designated 100 % survival. Samples were incubated for 1 day or 5 days before determining the c.f.u. ml−1 of each sample.

    RESULTS

    Deletion of hlp from M. smegmatis mc2155

    Deletion of the hlp gene was completed by allelic replacement with a HygR cassette. This replacement was confirmed by Southern blotting (Fig. 1a⇓). The labelled probe bound to a 2.125 kb band in the digested DNA from M. smegmatis mc2155 and a 3.537 kb band in the DNA from the mutant strain. The larger product in the mutant strain corresponds to an expected increase in size due to the replacement of the hlp gene with the HygR marker.

    Figure image not available in archive
    Fig. 1.

    Confirmation of hlp deletion in M. smegmatis. (a) Southern blot analysis shows that the probe for a DNA sequence flanking hlp binds to a 2.125 kb chromosomal fragment in the wild-type mc2155 strain (lane 1). For the Δhlp strain, this probe anneals to a fragment 1.4 kb larger, indicating the presence of the HygR cassette within hlp (lane 2). (b) PCR reveals that a 200 bp internal region of hlp present in mc2155 (lower band in lane 1) is absent from the Δhlp strain (lane 2). The upper band in lanes 1 and 2 is a non-specific PCR product that serves as an internal control. (c) Coomassie-stained 12 % SDS-PAGE of late exponential-phase cultures demonstrates elevated levels of Hlp protein (arrow) in mc2155 : : pMV306 (lane 1) and the complemented mutant strain Δhlp : : pMV306/hlp (lane 2), but not in Δhlp : : pMV306 (lane 3). An Invitrogen Benchmark pre-stained protein ladder was used as a molecular mass standard (lane M).

    PCR analysis showed the absence of an internal region of hlp in the chromosome of M. smegmatis Δhlp (Fig. 1b⇑). A non-specific PCR product (upper band) served as an internal control. Absence of an hlp-specific PCR product from M. smegmatis Δhlp corresponds with production of a HygR-specific PCR product that was not generated from mc2155 (data not shown). Loss of the Hlp protein was also demonstrated by SDS-PAGE (Fig. 1c⇑). Late exponential-phase cultures of M. smegmatis Δhlp : : pMV306 lacked an approximately 30 kDa protein (Fig. 1c⇑, lane 3) that was present in mc2155 : : pMV306 (lane 1) and Δhlp : : pMV306/hlp (lane 2). Despite its predicted size of 21 kDa, Hlp has been shown to migrate more slowly on SDS-PAGE due to its highly basic nature (Lee et al., 1998; Shires & Steyn, 2001).

    Loss of hlp alters cell and colony morphology

    Young colonies of the different strains showed marked differences in size. After 3 days growth, individual wild-type mc2155 colonies were much larger (Fig. 2a⇓, upper inset), with confluent colonies showing large ridges (Fig. 2a⇓, lower inset). Colonies of the Δhlp strain were much smaller and confluent colonies lacked prominent ridges (Fig. 2c⇓, upper and lower insets, respectively). As colonies aged, however, diameters of the two strains became more similar (compare Fig. 2b and d⇓). Two-week-old mc2155 colonies were rough in appearance, with ridges forming across the entire surface (Fig. 2b⇓). However, 2-week-old Δhlp colonies had a smoother, flatter morphology (Fig. 2d⇓). In addition, TEM analysis of 2-week-old cultures from agar surfaces showed that mc2155 cells were two- to threefold shorter than their Δhlp counterparts (compare Fig. 2b and d⇓, insets). The complemented Δhlp mutant had the typical rough colony appearance and shortened cell morphology of mc2155 (data not shown).

    Figure image not available in archive
    Fig. 2.

    Hlp affects colony and cell morphology. Strains mc2155 : : pMV306 (a) and Δhlp : : pMV306 (c) were grown for 3 days on 7H11 agar before visualization with a dissecting microscope. Insets show higher magnifications of individual colonies (upper insets) or of confluent colonies (lower insets). Bars, 1 mm. Single colonies of mc2155 : : pMV306 (b) and Δhlp : : pMV306 (d) were also allowed to continue growing for 2 weeks. TEM images (×5000) of 2-week-old cells are shown in the insets.

    Smooth colony phenotypes in M. smegmatis have been previously reported and linked with altered cell wall hydrophobicity (Arora et al., 2008; Chen et al., 2006). Without Tween 80, the hydrophobic nature of the M. smegmatis mc2155 cell wall causes the cultures to aggregate in liquid. When M. smegmatis Δhlp was grown in the absence of this detergent, the cells were able to grow dispersed in solution without the clumping that was seen in M. smegmatis mc2155 (results not shown).

    Growth and long-term survival of M. smegmatis Δhlp

    Growth of M. smegmatis Δhlp in 7H9+Tween 80 was similar to that of M. smegmatis mc2155 when grown at 37 or 45 °C (data not shown). Lee et al. (1998) examined the ability of an M. smegmatis hlp mutant strain to survive 2 weeks of bacterial dormancy by sustaining cultures in a low-oxygen environment. As mycobacteria are known to survive years of starvation (Sun & Zhang, 1999), we examined changes in long-term viability for up to 8 months. Loss of hlp had no effect on long-term viability in these 8-month-old, oxygen-starved cultures (results not shown).

    M. smegmatis Δhlp has increased sensitivity to UV exposure

    An earlier study showed that lack of the histone-like protein HU from E. coli leads to an increase in sensitivity to UV light (Li & Waters, 1998). This was due in part to an involvement in DNA repair processes (Miyabe et al., 2000). As a DNA-binding protein, Hlp could have an involvement in DNA repair, which might be demonstrated by alterations in UV sensitivity upon deletion of the hlp gene. Deletion of hlp from M. smegmatis did result in an increase in sensitivity to UV light (Fig. 3⇓). When exposed to up to 15 J m−2 UV, M. smegmatis Δhlp : : pMV306 had an eightfold decrease in survival compared with M. smegmatis mc2155 : : pMV306 or the complemented mutant strain Δhlp : : pMV306/hlp.

    Figure image not available in archive
    Fig. 3.

    Survival of M. smegmatis strains after exposure to UV light. Cell survival decreases with increased exposure to UV light. mc2155 : : pMV306 (□) and Δhlp : : pMV306/hlp (◊) have eightfold increased survival compared with Δhlp : : pMV306 (⧫). Error bars, sd for three samples.

    M. smegmatis Δhlp has increased sensitivity to INH

    Previous studies have linked alteration in cell wall composition and loss of a histone-like protein to alterations in antibiotic sensitivity (Arora et al., 2008; Colangeli et al., 2007; Jackson et al., 1999; Lewin et al., 2008). To determine whether loss of Hlp generates a similar effect on antibiotic sensitivity in M. smegmatis, isogenic strains were compared for sensitivity to various antibiotics. No differences were observed between the three strains for sensitivity to kanamycin, ethambutol or rifampicin (results not shown). However, M. smegmatis Δhlp : : pMV306 showed increased sensitivity to varying concentrations of INH. The zones of INH inhibition of M. smegmatis Δhlp were significantly larger than that of the wild-type or the complemented mutant strains (Fig. 4⇓). The MIC for INH of M. smegmatis Δhlp was determined to be 2.5 μg ml−1, which was fourfold smaller than that observed for isogenic strains containing a functional hlp gene.

    Figure image not available in archive
    Fig. 4.

    Loss of hlp confers increased susceptibility to INH. Zones of inhibition were measured around disks soaked with increasing concentrations of INH. Black bars, mc2155 : : pMV306; grey bars, Δhlp : : pMV306/hlp; white bars, Δhlp : : pMV306. Error bars, sd for three experiments.

    M. smegmatis Δhlp has increased sensitivity to freezing

    An earlier report linked hlp with the ability of M. smegmatis to adapt to a cold shock of 10 °C and resume growth (Shires & Steyn, 2001). While this study looked at the ability of an M. smegmatis hlp mutant to gradually acclimatize to a temperature of 10 °C, here we tested the ability of M. smegmatis Δhlp to survive a rapid temperature adjustment through repeated cycles of freezing and thawing (Fig. 5a⇓). After 21 rounds of repeated freezing and thawing, M. smegmatis Δhlp : : pMV306 had a 10-fold decrease in percentage survival compared with mc2155 : : pMV306 and Δhlp : : pMV306/hlp.

    Figure image not available in archive
    Fig. 5.

    Viability of M. smegmatis strains after repeated rounds of freezing and thawing. (a) Following 21 cycles of controlled freezing and thawing, viabilities of cultures were compared by determining c.f.u. ml−1. mc2155 : : pMV306 (black bar) and Δhlp : : pMV306/hlp (grey bar) have a 10-fold enhanced survival over Δhlp : : pMV306 (white bar). Cultures were submerged in a sphagnum moss bog for several months in spring and summer (b) or autumn and winter (c). Samples were removed at the designated months and compared for cell viabilities. □, mc2155 : : pMV306; ⧫, Δhlp : : pMV306; ◊, Δhlp : : pMV306/hlp. Error bars, sd for three samples. Average monthly surface temperatures are also shown (b, insert).

    Sphagnum peat bogs are environmental niches rich in mycobacterial diversity, and M. smegmatis can be found there (Kazda, 2000). Decaying sphagnum moss can act as a natural insulator to keep subsurface levels constant at 4–10 °C while daily surface temperatures fluctuate widely. During winter months, freezing can occur several centimetres downwards into the moss. Cryovial tubes containing different strains of M. smegmatis were incubated below the surface of sphagnum moss to let cultures be exposed to natural freezing and thawing cycles over a nine-month period. During the spring and summer months (March–July), there was no observed difference in survival of the three strains (Fig. 5b⇑). However, during the autumn and winter months, cells lacking hlp experienced as much as a 100-fold decrease in viability compared with cells expressing hlp (Fig. 5c⇑). This reduction in viability corresponds to surface and subsurface freezing temperatures. During the months of December 2009 and January 2010, average daily surface temperatures exceeded 0 °C only twice and not at all in February 2010.

    Survival at acidic pH

    Recently, Bi et al. (2009) showed that E. coli histone-like proteins (HU proteins) play an important role in allowing the bacteria to grow at low pH. To test whether M. smegmatis can survive at lower pH levels in an hlp-dependent fashion, isogenic strains were compared for viability after exposure to increasingly acidic media (Fig. 6⇓). Although a decreasing viability corresponded to decreasing pH levels (compare Fig. 6a, b and c⇓), there was no appreciable difference in acid tolerance between strains with or without hlp.

    Figure image not available in archive
    Fig. 6.

    Comparison of M. smegmatis strain survival during acid stress. Cultures of mc2155 : : pMV306 (□) and Δhlp : : pMV306 (⧫) were exposed to pH levels of 6.5 (a), 5.0 (b) and 4.0 (c) for increasing durations before testing for viability by measuring c.f.u. ml−1 and recording this as the percentage survival of cells with respect to time 0. Error bars, sd for three experiments.

    DISCUSSION

    In this report we have examined the role that hlp plays in stress tolerance and antibiotic sensitivity for M. smegmatis. Loss of Hlp adversely affects the ability of the organism to survive UV radiation (Fig. 3⇑) and freezing conditions (Fig. 5⇑). Additionally, M. smegmatis Δhlp was more susceptible to the antibiotic INH (Fig. 4⇑). However, the Hlp does not affect the growth rate of cells in liquid (Fig. 2a⇑), the ability to survive hypoxia and nutrient deprivation (Fig. 2b⇑), or the ability to withstand acidic conditions (Fig. 6⇑).

    Hlp is a small, basic, DNA-binding protein that was first characterized in M. smegmatis as a major protein induced in response to cellular dormancy (Lee et al., 1998). Hlp shares similarity to other prokaryotic histone-like proteins through its N-terminal domain, and to the eukaryotic histone H1 proteins through its C-terminal domain (Prabhakar et al., 1998). As a histone-like protein, Hlp might play roles in DNA compaction and gene regulation necessary for stress survival. The ability of Hlp to act as a transcriptional repressor in M. smegmatis in vitro means that it may regulate gene activity (Mukherjee et al., 2008), although at present the full significance of Hlp in M. smegmatis has not been elucidated. Hlp homologues have been found localized to the cytosol and nucleoid (Mukherjee et al., 2008; Prabhakar et al., 1998), and to the cell wall (Aoki et al., 2004; Soares de Lima et al., 2005; Yeruva et al., 2006). This localization suggests a complex and multifunctional role for these structurally unique mycobacterial histone-like proteins.

    We have recently reported that loss of the histone-like protein Lsr2 from M. smegmatis produces a smooth colony (Arora et al., 2008; Chen et al., 2006). The deletion of hlp from the M. smegmatis chromosome also results in a smoother colony morphology (Fig. 2c, d⇑). This suggests changes in the cell envelope composition. Using scanning electron microscopy, Katsube et al. (2007) showed that stationary-phase cells of an M. smegmatis hlp mutant had crenellated surfaces, unlike the smooth cell surface seen in wild-type M. smegmatis. These authors also demonstrated that Hlp was involved in the downregulation of cell surface components in stationary phase (Katsube et al., 2007). These results all appear to confirm that loss of Hlp affects the composition of the cell envelope. Such changes often coincide with differences in cell hydrophobicity (Chen et al., 2006; Ojha et al., 2005). A decrease in the hydrophobicity of M. smegmatis Δhlp was evident from its ability to grow dispersed in liquid media without the addition of the detergent Tween 80 (results not shown). Given that Hlp homologues are found localized to the cell envelope (Katsube et al., 2007; Soares de Lima et al., 2005) and that Hlp can bind externally to Mycobacterium bovis (Katsube et al., 2007), it is possible that Hlp is involved in cell-to-cell aggregation of M. smegmatis. Lewin et al. (2008) found that reducing expression of the Hlp homologue in M. bovis decreased cell aggregation.

    Because hlp is upregulated in response to bacterial dormancy, Lee et al. (1998) examined the effect of the loss of Hlp on the short-term viability of oxygen-starved cultures left stationary for up to 2 weeks. While they did not observe any decrease in viability without hlp, M. smegmatis can exist in a dormant state for years. Therefore, we repeated this experiment for up to 8 months but were unable to observe any effect on long-term viability in oxygen-starved cultures due to loss of hlp (results not shown). Lewin et al. (2008) have reported that there is no correlation between Hlp levels and growth rates of M. bovis under low-oxygen conditions. However, differences in protein production were observed if Hlp levels were reduced, suggesting that Hlp plays a role in protein expression of M. bovis BCG in hypoxic conditions. Further work is needed to determine whether the loss of Hlp in M. smegmatis affects gene expression under low oxygen.

    M. smegmatis Δhlp had an eightfold decrease in survival when subjected to UV light (Fig. 3⇑). This is in contrast to an M. smegmatis Δsmc mutant, which shows no change in levels of repair of UV-induced lesions (Güthlein et al., 2008). This is despite the role of SMC (structural maintenance of chromosomes) proteins in repair of DNA damage in different species of bacteria. In E. coli, deletion of the heterodimer formed by the histone-like proteins HUα and HUβ leads to an over 100-fold decrease in UV survival (Li & Waters, 1998). This susceptibility to UV light in E. coli is attributed to a deficiency in RecA-dependent DNA repair and SOS induction (Miyabe et al., 2000). Although Hlp appears to play less of a protective role in M. smegmatis than in E. coli (eightfold compared with 100-fold), it is possible that Hlp of M. smegmatis is also involved in DNA repair. A recent publication has demonstrated that Hlp enhances DNA end-joining in M. smegmatis in vitro (Mukherjee et al., 2008). An additional reason for the increased susceptibility to UV seen here in the Δhlp strain could be the alteration in the cell envelope structure. As a major barrier against the environment, changes in the cell envelope could affect UV sensitivity. For example, the loss of genes involved in cell envelope composition in Lactococcus lactis leads to an increase in UV sensitivity (Duwat et al., 1997).

    M. smegmatis Δhlp was tested for its susceptibility to four antibiotics: INH, rifampicin, ethambutol and kanamycin. No change in the susceptibility to rifampicin, ethambutol or kanamycin was observed. This result is similar to that seen in M. bovis, where reduced expression of the Hlp homologue has no affect on the rifampicin susceptibility (Lewin et al., 2008). The loss of Hlp from M. smegmatis did lead to a fourfold increase in susceptibility to INH (Fig. 4⇑). INH is a front-line drug for the treatment of tuberculosis, and is a small hydrophilic prodrug that enters into the cell by passive diffusion, where it is activated by the action of KatG (Zhang & Telenti, 2000). Activated INH can inhibit enzymes involved in the synthesis of mycolic acids (Zhang & Telenti, 2000). Further cellular damage can occur as a result of the action of reactive oxygen radicals, which are produced during prodrug activation (Zhang et al., 1996). With the loss of Hlp, M. smegmatis may be more susceptible to the free radicals produced by INH activation, suggesting an involvement of Hlp in the resistance of M. smegmatis to oxidative damage. It is possible that alterations in the cell wall of M. smegmatis Δhlp increase cell permeability to INH. However, this does not seem likely, since no Hlp-dependent differences were seen in the sensitivities to the other antibiotics tested, including ethambutol, a similarly small hydrophilic antibiotic.

    Tolerance to freeze–thaw activity is a complex mechanism involving the accumulation of trehalose, the production of molecular chaperones, and the composition of the bacterial cell wall (Tanghe et al., 2003). M. smegmatis Δhlp has decreased tolerance to repeated freezing and thawing (Fig. 5⇑). An earlier study demonstrated the inability of an M. smegmatis hlp mutant to resume growth after cultures were shifted from 37 to 10 °C (Shires & Steyn, 2001). This study examined cellular viability after repeated rounds of freezing and thawing. As a major cold-shock-inducible protein, Hlp could be involved in stabilizing mRNA transcripts and allowing transcription initiation to occur after a cold shock (Shires & Steyn, 2001). However, the method used here suggests a different role for Hlp in freeze tolerance as the cells were rapidly shifted to a state of metabolic inactivity (Fig. 5a⇑). In lactobacilli, the fatty acid composition of the cell membrane has been linked to the tolerance of freezing and thawing (Gomez Zavaglia et al., 2000), and it is possible that the altered composition of the M. smegmatis Δhlp cell envelope lowers freeze tolerance.

    Attempts were made in this study to test in the laboratory the conditions that M. smegmatis would naturally encounter in a peat bog. Sphagnum moss bogs are acidic, oligotrophic, anoxic environments with poor microbial growth. Typically these environments are located in boreal and arctic regions subject to periodic annual snow cover. Although these environments provide numerous physical hindrances to microbial growth, mycobacteria are found in great abundance and diversity in the slowly decaying dead layer (grey–brown layer) several centimetres below the green, actively growing surface of the moss. Mycobacteria are not present in the green canopy of the moss because it is extremely acidic as a result of cation antiport, in which moss cells export protons in order to take up essential cations (Brennan & Nikaido, 1995). Hpl does not appear necessary for M. smegmatis to tolerate low oxygen, low nutrients or low pH, but Hpl is likely to help M. smegmatis withstand repeated bouts of freezing as well as penetrating UV light.

    The pleiotropic phenotype of the hlp mutant suggests that some regulator mechanism has been altered. It is possible that alterations in a single cell envelope moiety could be responsible for all of the phenotypes reported here, but this seems less feasible than changes in the regulation of multiple genes due to loss of Hlp. Further study of this possibility will be conducted through microarray comparisons of the wild-type and Δhlp strains.

    Acknowledgments

    We thank Christine Davitt for assistance with TEM analysis. This research was supported by internal funds from the University of Minnesota Duluth.

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