A lipoprotein modulates activity of the MtrAB two-component system to provide intrinsic multidrug...

A lipoprotein modulates activity of the MtrAB two-componentsystem to provide intrinsic multidrug resistance, cytokineticcontrol and cell wall homeostasis in Mycobacteriummmi_7110 348..364

Hoa T. Nguyen,† Kerstin A. Wolff,†

Richard H. Cartabuke, Sam Ogwang andLiem Nguyen*Department of Molecular Biology and Microbiology,School of Medicine, Case Western Reserve University,Cleveland, Ohio 44106, USA.

Summary

The MtrAB signal transduction system, which partici-pates in multiple cellular processes related to growthand cell wall homeostasis, is the only two-componentsystem known to be essential in Mycobacterium. In ascreen for antibiotic resistance determinants in Myco-bacterium smegmatis, we identified a multidrug-sensitive mutant with a transposon insertion in lpqB,the gene located immediately downstream of mtrA–mtrB. The lpqB mutant exhibited increased cell–cellaggregation and severe defects in surface motilityand biofilm growth. lpqB cells displayed hyphalgrowth and polyploidism, reminiscent of the morphol-ogy of Streptomyces, a related group of filamentousActinobacteria. Heterologous expression of M. tuber-culosis LpqB restored wild-type characteristics to thelpqB mutant. LpqB interacts with the extracellulardomain of MtrB, and influences MtrA phosphorylationand promoter activity of dnaA, an MtrA-regulatedgene that affects cell division. Furthermore, in transexpression of the non-phosphorylated, inactive formof MtrA in wild-type M. smegmatis resulted in pheno-types similar to those of lpqB deletion, whereasexpression of the constitutively active form of MtrArestored wild-type characteristics to the lpqB mutant.These results support a model in which LpqB, MtrBand MtrA form a three-component system thatco-ordinates cytokinetic and cell wall homeostaticprocesses.

Introduction

The primary machineries that help bacteria sense andrespond precisely to surrounding environments are two-component signal transduction systems. A classical two-component system consists of a transmembrane sensorhistidine kinase (HK) and a cytoplasmic response regula-tor (RR) protein. Upon recognizing certain stimuli, thesensor kinase autophosphorylates one of the histidineresidues in its cytoplasmic domain, which then transfersthe phosphate group to an aspartate residue of thecognate RR. The phosphorylated RR then becomesactive and modulates expression of its regulon (genesregulated by the RR) leading to alterations of cell behav-iours in response to the stimuli. The genes encoding theHK and its cognate RR are often genetically linked andtherefore classically named ‘two-component’ system.

Multiple-component systems have also been reportedin which auxiliary proteins assist or modify activities of thecore two-component systems. A typical example of suchcomprehensive multiple-component transduction systemsis the YycFG system (also called VicRK, MicBA or WalRKin different bacteria) that is highly conserved in low G+CGram-positive bacteria (Firmicutes) (Szurmant et al.,2007a; Dubrac et al., 2008; Winkler and Hoch, 2008).YycFG controls several cellular pathways involved in cellwall synthesis, cell growth and cell division (Fabret andHoch, 1998; Ng et al., 2004; 2005; Mohedano et al., 2005;Bisicchia et al., 2007; Dubrac et al., 2007; Fukushimaet al., 2008). Mutations in this system affect multiple pro-cesses such as cell wall permeability, antibiotic resis-tance, lipid integrity, biofilm formation, cellularmorphology, osmotic stress, as well as virulence of patho-genic bacteria (Martin et al., 1999; Wagner et al., 2002;Mohedano et al., 2005; Senadheera et al., 2005; Liuet al., 2006; Deng et al., 2007; Dubrac et al., 2007;Jansen et al., 2007; Senadheera et al., 2007). Toco-ordinate such a diverse array of functions, YycFGcross-talks with other two-component systems (Howellet al., 2006), and integrates signals from multiple acces-sory proteins. In all bacteria having this system, the genesencoding the HK YycG and the RR YycF are cotrans-cribed with genes encoding their accessory proteins,

Accepted 15 February, 2010. *For correspondence. E-mail liem.nguyen@case.edu; Tel. (+1) 216 368 3148; Fax (+1) 216 368 3055.†These authors contributed equally to this work.

Molecular Microbiology (2010) 76(2), 348–364 � doi:10.1111/j.1365-2958.2010.07110.xFirst published online 31 March 2010

YycH, YycI and YycJ. Whereas YycJ (called VicX in Strep-tococcus) modulates functions of YycFG in biofilm forma-tion, genetic competence and oxidative stress resistance(Senadheera et al., 2007), the transmembrane domainsof YycH and YycI were shown to modulate the autophos-phorylation status of the HK YycG (Szurmant et al., 2005;2007b; 2008).

In high G+C Gram-positive bacteria (Actinobacteria),the functional analogue of the YycFG system has beensuggested to be the MtrAB two-component system (Hosk-isson and Hutchings, 2006; Winkler and Hoch, 2008).Similar to YycFG in Bacillus subtilis, MtrAB is the onlytwo-component system known to be essential in Myco-bacterium (Zahrt and Deretic, 2000; Rison et al., 2005;Robertson et al., 2007), and alterations in MtrAB expres-sion also lead to cell wall and cell division homeostaticdefects such as altered cell morphology, reduced antibi-otic resistance and attenuated virulence (Möker et al.,2004; Cangelosi et al., 2006; Fol et al., 2006). The mtrABlocus was first identified in M. tuberculosis by heterolo-gous hybridization with a Pseudomonas aeruginosa phoBprobe (Via et al., 1996). This locus is highly conserved inActinobacterial genomes (Fig. S1). Transcription of theRR-encoding gene mtrA, which is controlled by the viru-lence sigma factor C (SigC) (Sun et al., 2004), is inducedin M. bovis BCG during macrophage infection, but itsexpression is constitutive in M. tuberculosis (Dhand-ayuthapani et al., 1995; Via et al., 1996). Overexpressionof mtrA leads to increased expression of DnaA, themaster regulator of DNA replication, and inhibition of M.tuberculosis proliferation in human macrophages (Folet al., 2006). In Corynebacterium, mtrAB is not essential(Möker et al., 2004), and its function is also related to pHcontrol, osmotic protection, as well as cold shockresponse (Möker et al., 2004; 2006; 2007).

Accessory proteins of the MtrAB system have not beenidentified. Based on chromosomal localization, it wasspeculated that the putative lipoprotein LpqB, whoseencoding gene is located immediately downstream ofmtrA and mtrB, might act as a modulator of MtrB activity(Hoskisson and Hutchings, 2006). Despite this specula-tion, the function of LpqB is completely unknown. Here wereport the characterization of the first lpqB mutant, whichwas unexpectedly isolated in a screen for multidrug-sensitive mutants of Mycobacterium smegmatis. Disrup-tion of lpqB resulted in pleiotropic effects includingincreased multidrug susceptibility, retarded surface motil-ity and biofilm growth, as well as defects in morphologicaland cell division control. Our experiments showed thatLpqB interacts with the extracellular domain of MtrB andaffects the MtrA phosphorylation status that mediatesalterations in cell division and cell wall homeostasis. Fur-thermore, lpqB disruption alters expression of dnaA, anMtrA-regulated gene that controls DNA replication. Col-

lectively, the results support a model in which LpqB func-tions as an accessory protein that modulates activities ofthe Actinobacterial two-component system MtrAB toprovide antibiotic resistance, cell division and cell wallhomeostatic control.

Results

Identification of LpqB – a novel determinant of multidrugresistance in M. smegmatis

A library of ~7000 transposon mutants was generatedfrom wild-type M. smegmatis mc2155 by Himar1-mediatedmutagenesis, and deposited in 96-well plates. This librarywas used to screen for mutants with increased suscepti-bility to antibiotics (Nguyen et al., 2005; Wolff et al., 2009).A subgroup of these drug-sensitive mutants, which dis-played Multiple Antibiotic Resistance defective pheno-types, was designated as the MARs.

A MAR mutant, MAR2, found in a screen forerythromycin- and vancomycin-sensitive mutants, exhib-ited additional susceptibility to other classes of antibioticswith diverse chemical structures and functions. Theseinclude antibiotics targeting several steps in the cell wallbiosynthetic pathways, such as those of the carbapenemsubgroup of b-lactam antibiotics, vancomycin and bacitra-cin; antibiotics targeting protein synthesis, such as thoseof the macrolide, streptogramin and aminoglycosideclasses; antibiotics targeting DNA replication such asnovobiocin; as well as those targeting transcription, suchas rifampicin which is also a front-line tuberculosis drug(Fig. S2, Table S1). Minimum inhibitory concentrations(MICs) for five representative antibiotics, quantified byusing E-test assay (AB Biodisk, Sweden) as previouslydescribed (Nguyen et al., 2005; Wolff et al., 2009), indi-cated that MAR2 was 63-, 1000-, 16-, 80- and 6-fold moresusceptible than the wild-type strain to vancomycin, eryth-romycin, imipenem, rifampicin and amoxicillin/clavulanicacid respectively (Table 1).

Genetic mapping using a nested PCR method as pre-viously described (Nguyen et al., 2005) localized thetransposon insertion to a gene encoding a putative lipo-protein of unknown function (msmeg_1876) (Fig. 1). The

Table 1. Susceptibility of M. smegmatis strains to antibiotics.

Strain

MIC (mg ml-1)

VA EM IP RI XL

mc2155 24 96 2 4 6MAR2 0.38 0.096 0.125 0.049 1MAR2/lpqBTB 8 2 0.25 3 2

VA, vancomycin; EM, erythromycin; IP, imipenem; RI, rifampicin; XL,amoxicillin/clavulanic acid.

Multidrug resistance in mycobacteria. 349

deduced amino acid sequence of the mutated genehad highest similarities to a hypothetical lipoprotein inM. tuberculosis (LpqBTB) and homologues in othermycobacteria. In all available Actinobacterial genomesequences where homologues of LpqB were found, theencoding genes were always clustered with two othergenes, mtrA and mtrB, which, respectively, encode for aRR (MtrA) and a HK (MtrB) of a signal transductionsystem (Fig. S1).

The Himar1 insertion in the lpqB gene of M. smegmatisMAR2 was further confirmed by PCR amplification of the

mutant locus, using primers flanking the putative openreading frame. The mutant gene generated a larger frag-ment corresponding to the inserted transposon (2199 bp).Insertion of the transposon resulted in a decreased mobil-ity of the PCR product on an agarose gel (Fig. 1B).Sequencing of the lpqB-Himar junction region from thisPCR product identified the insertion site at the dinucle-otide TA517–518 that introduced a stop codon after the tripletencoding the Leu172 residue (Fig. 1A, top). To confirmloss of the lpqB gene product, Western blots were doneusing a polyclonal antibody raised against a synthetic

mc2 15

lpqB ag84

mc2 15

0.60.81.0

1.52.02.53.04.05.0

lpqB+himar1

M. smegmatis mc2155

mtrB mtrA lpqB

517-518

M. smegmatis MAR2 himar1STOPlpqB

tgg cag cag ttc cag gcc acc tac aag cgc tac acc ctc ta A C A G G T T G G

W Q Q F Q A T Y K R Y T L*

100150250

LpqB Ag84

Fig. 1. Identification of MAR2 – an lpqB transposon mutant.A. Himar1 transposon insertion in a gene encoding a putative lipoprotein termed LpqB in MAR2. Sequencing revealed the Himar1 insertion atthe dinucleotide TA517–518 that introduced a stop codon after the triplet encoding Leu172 residue. Bar, 1 kb.B. PCR amplification using primers (arrows shown in Fig. 1A) flanking the lpqB open reading frame of wild-type M. smegmatis mc2155. Thetransposon insertion in MAR2 resulted in a corresponding increase in the size of the PCR product. As a control, PCR amplification of the geneencoding for the antigen 84 (ag84, msmeg_4217) produced identical products from both wild-type M. smegmatis and MAR2 genomic DNA.The same molecular weight markers were run on the left of each agarose gel.C. Western blot using anti-LpqB antibody. LpqB was detected in cell lysates of wild-type M. smegmatis but undetectable in the extracts ofMAR2. Transformation of plasmid pVN751 resulted in heterologous expression of M. tuberculosis LpqB in MAR2. As a control, Ag84 wasequally detected in all M. smegmatis strains.All data are representative of at least two independent experiments from biological replicates.

350 H. T. Nguyen et al. �

peptide sequence of LpqB (see Experimentalprocedures). The antibody recognized a protein band of~61 kDa corresponding to the predicted molecular weightof LpqB in the cell lysate of wild-type M. smegmatis, whichis absent in the lysate of MAR2, confirming the absence ofLpqB expression in MAR2 (Fig. 1C).

Cross-species expression of M. tuberculosis LpqBrestores wild-type multidrug resistance to MAR2

To confirm that the multidrug sensitivity phenotype ofMAR2 was due to a lack of LpqB activity, the lpqB gene ofM. tuberculosis (lpqBTB) was cloned and expressed in theMAR2 mutant. Transformation of the pVN751 [PSOD-lpqBTB]plasmid (Table S2) resulted in restoration of LpqB expres-sion in MAR2 (Fig. 1C). Heterologous expression ofLpqBTB (76% identity and 86% similarity to M. smegmatisLpqB) increased drug resistance of MAR2 (Table 1), indi-cating that the increased antibiotic susceptibility of MAR2was due to the lpqB deletion. The result also suggests thatLpqB provides this function not only to M. smegmatis butalso to other mycobacterial species. However, the LpqBproteins from the two bacterial species might carry somelevels of specificity because complementation was notcomplete for some antibiotics (Table 1).

Plasmid pVN751 produced an increased level of LpqB(Fig. 1C). However, this increased expression did not leadto an increased drug resistance (Table 1), suggesting thatLpqB does not directly control dedicated drug resistancemechanisms; but rather that its deletion affects principalcellular processes (e.g. cell wall biosynthesis or homeo-stasis) that influence drug susceptibility. In fact, the over-expression of LpqB from pVN751 per se might have alsocaused the incomplete complementation of MAR2 due toits reverse effects on downstream gene expressionleading to disorders in such cellular processes.

Disruption of LpqB results in altered colonialmorphology and cell aggregation

The MAR2 mutant displayed abnormal morphology atseveral levels. In liquid media, MAR2 culture exhibited anincreased cell–cell aggregation. After 2 days of growth in7H9, the wild-type culture was still well suspended in themedium whereas the MAR2 culture was readily settled atthe bottom of culture tubes (Fig. 2A). This reflected analteration in cell wall composition, surface properties orcellular morphology of MAR2 cells. Altered cell–cell inter-actions or cell wall surface properties usually give rise tochanges in colonial morphology. On solid media, coloniesof MAR2 showed a caved-in structure without densecores (Fig. 2B). This altered colonial morphology wasmost obvious on 7H10 medium supplemented withTween-80. Although they were not exactly like wild-type,

colonies of the MAR2 strain expressing M. tuberculosisLpqB lost the caved-in structure, characteristic of MAR2morphology (Fig. 2B, right panel).

Morphological changes associated with increased mul-tidrug sensitivity of the M. avium mtrB mutant correlateswith increased Congo Red staining (Cangelosi et al.,2006). In addition, M. avium clinical isolates with anincreased Congo Red uptake (red morphotype) alsodisplay reduced multidrug resistance and virulence com-pared with isogenic ‘white’ counterparts (Cangelosi et al.,2001). After 4 days of growth on a solid medium containingCongo Red at 37°C (without Tween-80), a clear phenotypicdifference among the M. smegmatis strains was observed,which correlated with LpqB expression. In contrast to thepink and dry colonies of wild-type and the complementedstrain, MAR2 colonies were red with shiny wet cores(Fig. 2C), indicating an increased Congo Red uptake. Insupport of a defective cell wall homeostasis, MAR2 alsoexhibited an increased sensitivity to the detergent sodiumdodecyl sulphate (SDS) when grown on a solid medium.MAR2 was at least 100-fold more susceptible to SDS thanwild-type (Fig. 2D). All wild-type morphological character-istics were restored when MAR was complemented withplasmid pVN751 expressing M. tuberculosis LpqB.

Disruption of LpqB affects surface motility andbiofilm formation

Because the MAR2 mutant showed an abnormal colonialmorphology and cell–cell aggregation, we determinedwhether LpqB is also required for sliding motility andbiofilm formation. As expected, MAR2 was defective insliding on the surface of minimum medium agar plates,and this defect could be complemented by expression oflpqBTB (Fig. 3A). Studies of M. smegmatis biofilms haveestablished a relationship between surface motility andthe ability to form biofilms (Recht and Kolter, 2001; Aroraet al., 2008). Wild-type M. smegmatis, MAR2 and thecomplemented strain were subjected to an air-liquidbiofilm assay (Ojha et al., 2005). At day 3 after inocula-tion, the culture of wild-type M. smegmatis had alreadycolonized the whole surface of the liquid medium,whereas the MAR2 culture only formed unconnectedclumps in the liquid phase and a few islands of growth onthe surface. At day 5, typical mature biofilm structuresappeared on the surface of the wild-type culture, but theformation of those structures was clearly delayed on thesurface of MAR2 (Fig. 3B and C).

Disruption of LpqB affects cell division

Microscopic analyses revealed a striking phenotype of theMAR2 mutant. Instead of the normal rod shape of wild-type M. smegmatis cells, MAR2 cells grew as branched

filaments (Fig. 4A, white arrows). The length of MAR2cells was significantly greater than that of wild-type withmany cells reaching 20 mm (Fig. 4A and B; Fig. S3).Cross-species expression of lpqBTB restored normal celllength to MAR2 (Fig. 4A, right panel). For quantitativeanalysis, the length of 100 random cells of wild-type andMAR2 was measured after 24 and 51 h of growth in aliquid medium (Fig. 4B). Whereas wild-type cells had an

average cell length limited to a 3–5 mm range at both timepoints, MAR2 cells exhibited a wide range of lengths, from3 to 20 mm (Fig. 4B). After 51 h, 75% of MAR2 cells weregreater than 8 mm in length. These observations sug-gested that MAR2 has a defect in cell length or divisioncontrol.

Cell division control is commonly coupled to DNA rep-lication and segregation. To investigate nucleoid local-

mc2 15

- SDS + SDS

mc2155 MAR2 MAR2/ pVN751

ization in the filamentous MAR2 cells, they were stainedwith SYTO 11, a membrane permeable dye that fluo-resces when bound to nucleic acids. Whereas wild-typeM. smegmatis displayed mostly 1–2 fluorescent seg-ments per cell, MAR2 filaments fluoresced in a beadedpattern along the cell length, suggesting that they carrymultiple chromosomes (Fig. 4A). Most filaments ofMAR2 cells contained from 2 up to 12 fluorescent beadswhen examined at 24 and 51 h post inoculation(Fig. 4C). It is noteworthy that some of the filamentscontained empty sections (Fig. 4A, empty arrows),similar to the effect caused by depletion of yycF in B.subtilis (Fabret and Hoch, 1998). Strikingly, 42% and50% of the MAR2 filaments examined at these time

points had branches respectively. These branched com-partments, commonly budded out at loci of putativesepta, also contained nucleoids (Fig. 4A, white arrows).This filamentous, branched, polyploid morphologyclosely resembled Streptomyces, another genus belong-ing to the taxon Actinobacteria.

lpqB encodes a membrane-bound lipoprotein

Protein sequences of LpqB orthologues revealed thepresence of a signal peptide typically present at theN-terminus of lipoproteins, which is required for secretionthrough the general Sec secretion system (Fig. 5A). Todetermine the subcellular localization of LpqB, cell frac-

Fig. 2. Effects of LpqB expression on cell wall characteristics.A. The MAR2 liquid culture displayed a drastically enhanced cell aggregation. Cultures of M. smegmatis strains were grown for 48 h at 37°Cin 7H9 medium. Pictures of standing cultures were taken 5 min after removal from a shaking incubator. Expression of M. tuberculosis LpqBrestored wild-type aggregation level to MAR2.B. Colonial morphology of wild-type M. smegmatis, MAR2, and the MAR2 expressing M. tuberculosis LpqB grown on 7H10 mediumsupplemented with 0.5% Tween 80 for 3 days at 37°C. Expression of M. tuberculosis LpqB abolished the caved-in morphology of MAR2colonies.C. Congo red stain of M. smegmatis strains. Cultures (10 ml) of wild-type M. smegmatis, MAR2 and the MAR2 expressing M. tuberculosisLpqB were dropped on the surface of 7H9-agar plates containing 100 mg ml-1 Congo red. MAR2 cultures showed a distinctive wet, shiningcore and increased Congo red stain. Expression of M. tuberculosis LpqB restored wild-type colonial morphology to MAR2.D. Sensitivity of M. smegmatis strains to SDS. Ten-fold serial dilution of mycobacterial cultures (10 ml, starting OD ~0.2) were spotted on NEmedium containing no SDS (left) or 0.001% SDS (right) and incubated at 37°C. Growth was recorded at day 3 after inoculation.All data are representative of at least two independent experiments from biological replicates.

Fig. 3. Effects of LpqB on surface motilityand biofilm growth.A. Sliding motility of M. smegmatis strains onM63 medium. M. smegmatis strains weregrown in liquid 7H9 medium to mid log phase,spotted on M63 medium containing 0.3% agarand grown at 37°C. Representative coloniesat day 5 after inoculation are presented in thelower panel. Diameter (cm) of the colonies atday 5 was measured and presented in thegraph (upper panel). The determination wasperformed in biological triplicates. Error barsrepresent standard deviations.B. Surface biofilm growth of M. smegmatisstrains. Static cultures of M. smegmatisstrains were grown in polysterene Petri platescontaining biofilm medium at 30°C andpictured everyday. MAR2 displayed delays inboth the initial phase when cells grewtogether to form immature thin films and thematuration phase when typical wrinklestructures were formed on the surface. Shownare representative biofilms recorded at day 5after inoculation. The insets showstereomicroscopic observation (15¥) of thebiofilm surface.C. Estimation of biofilm growth ofM. smegmatis strains. Biomass from thesurface growth was isolated and total proteincontent was measured using Bradford assay.MAR2 showed a dramatic reduction of biofilmgrowth in this condition. The values are themeans of biological triplicates. Error barsrepresent standard deviations.

mc2155 MAR2 MAR2/ pVN751

mc2 15

in / p

Motilit

mc2 15

tionation was carried out, followed by Western blot analy-ses with LpqB antibody. In both M. smegmatis and thepathogenic M. tuberculosis (Fig. 5B, left and right panelsrespectively), LpqB was predominantly present in the cellmembrane and cell wall fractions, suggesting that LpqB

function is associated with its localization in membrane orcell wall. This was further supported by the fact thatexpression of a cytoplasmic form of LpqB failed to restorewild-type phenotypes to the MAR2 mutant (not shown).Next, to validate the lipoprotein nature of LpqB, M. smeg-

Fig. 4. Role of LpqB in cellular morphology,length and ploidy.A. DIC and fluorescence microscopy analysis.Nucleoids were visualized by the nucleic acidstain SYTO 11. Bacteria were grown in 7H9medium and stained for 30 min in 30 mMSYTO 11. Cells were washed twice with PBS,placed on agarose pads and observed underDIC (upper panels) or fluorescence (lowerpanels) filter. In contrast to the normal rodshaped, and mono- or dinucleoid morphologyof wild-typeM. smegmatis, MAR2 cells were filamentous,polyploid and branched (white arrows). SomeMAR2 filaments contained empty sections(empty arrows). In trans expression ofM. tuberculosis LpqB restored wild-typemorphology to MAR2. Bar, 5 mm.B. Length of wild-type M. smegmatis (circle)and MAR2 (triangle) measured at 24 and 51 hafter inoculation. Bacteria were grown in 7H9medium at 37°C. At each time point, length of~100 cells was measured and classified intogroups of �0.5 mm. Percentage was plottedagainst cell length.C. Ploidy of MAR2 cells analysed at 24 and51 h post inoculation. SYTO 11 stained cells(~100) were observed under a fluorescencemicroscope and the number of fluorescentbeads per cell were recorded. Cells wereclassified into ploidy groups and theirfrequency (%) was plotted.

1 2 3 4 5 6 7 8 9 10 11 12

0 2 4 6 8 10 12 14 16 18 20 22

Cell length (μm)

0 2 4 6 8 10 12 14 16 18 20 22

Cell length (μm)

24 hours 51 hours

MAR2mc2155 MAR2mc2155

Ploidy (beads/cell)

MAR2MAR2

C 24 hours 51 hours

A mc2155 MAR2/ pVN751D

matis strains expressing LpqB were grown in the pres-ence of globomycin, a specific inhibitor of the signalpeptidase LspA (Inukai et al., 1978; 1979) required for theremoval of signal peptides from lipoproteins after theirtranslocation to the extracytoplamic milieu. A mobility shiftof lipoproteins on SDS-PAGE gels usually indicates theinhibitory effect of globomycin on the removal of signalpeptides (Gibbons et al., 2007). When wild-type M. smeg-matis or the MAR2 strain overproducing LpqBTB wasgrown in the absence of globomycin and the proteinextracts were separated in the conditions described inExperimental procedures, LpqB was detected as twobands migrating closely to each other [Fig. 5C (-)].However, when the bacterial cultures were treated withglobomycin, a clear shift in mobility was observed for thelower band [Fig. 5C (+)], suggesting that the signal

peptide processing of LpqB was inhibited. Collectively,these results suggest that LpqB is a lipoprotein that ispresent in the cytoplasmic membrane and cell wall of themycobacterial cells.

LpqB interacts with the extracellular domain of MtrB

Phenotypic similarities of MAR2 and mutants of the adja-cent genes mtrA and mtrB (Figs 2–4; Möker et al., 2004;Brocker and Bott, 2006; Cangelosi et al., 2006; Fol et al.,2006) suggested that these genes might work together inthe same pathway(s). The presence of LpqB in the cyto-plasmic membranes and cell wall of M. smegmatis (Fig. 5)further suggested that LpqB activities were more directlyrelated to the membrane-bound MtrB, than the cytoplasmicMtrA. To investigate if LpqB interacts with MtrB, their in vivo

LpqBMS

LpqBTB

Signal peptide

Lipobox

Pre-LpqB

mc2 15

- + - +

T CW MS

LpqHM. s

isT CW MS

LpqHM. t

Fig. 5. Subcellular localization and lipoprotein nature of LpqB.A. Alignment of the first 40 N-terminal amino acid sequences deduced from the nucleotide sequences of M. smegmatis and M. tuberculosislpqB genes. Darker background shading indicates a lower degree of conservation. The amino acid sequences of the putative signal peptidesare underlined. Sequences resembling the lipoprotein box are indicated by a solid-lined box. The arrow indicates the cysteine residues towhich a diacylglycerol residue is ligated during lipoprotein processing. Signal peptide predictions were made using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP) and SIG-Pred (http://www.bioinformatics.leeds.ac.uk/prot_analysis/Signal.html).B. Subcellular fractionation of M. smegmatis (left panel) or M. tuberculosis cells (right panel) and detection of LpqB. Wild-type M. smegmatiswas subjected to subcellular fractionation by differential centrifugation. Equal amounts of total protein from total cell lysate (T), cell wall (CW),soluble fraction (S) and membrane (M) fractions were separated on SDS-PAGE, followed by Western blot using antibodies of LpqB, Ag84(F126-2) and LpqH (IT-19). M. tuberculosis subcellular fractions were obtained from Colorado State University.C. Inhibition of the signal peptidase activity on LpqB. Cultures of M. smegmatis strains were incubated in the absence (-) or presence (+) ofglobomycin that inhibits the lipoprotein signal peptidase (LspA). Mobility of proteins on SDS-PAGE gels was extended to allow separation ofLpqB forms (Experimental procedures). Two bands of LpqB were observed in cultures of non-treated M. smegmatis whereas only the upperband was detected in cultures treated with globomycin.All data are representative of at least two independent experiments from biological replicates.

association was investigated using a bacterial two-hybridsystem (Bacteriomatch, Stratagene). Different domains ofMtrB (Fig. 6A, and the far left panel in Fig. 6B) were clonedand expressed as fusions to the C-terminal end of thefull-length lcI protein in the bait vector pBT. A cytoplasmicpeptide of LpqB (LpqB22–583, without the signal peptide) wasfused to the N-terminal domain of the a-subunit of RNApolymerase in the target vector pTRG. Bacteriomatch IReporter strain (Stratagene) was co-transformed with

these fusion constructs. An interaction between bait andtarget peptides would recruit RNA polymerase to the pro-moter and activate the transcription of the reporter genesamp R and lacZ that allow growth in the presence ofcarbenicillin and the production of b-galactosidase respec-tively. Out of all the combinations of the plasmids (Fig. 6B,first and second panels from left), only combination 2(positive control, Gal11p + LGF2) and combination 6(MtrB140–214 + LpqB22–583) produced colonies of the reporter

Fig. 6. Interaction of LpqB and the cognatehistidine kinase MtrB.A. Two possible topologies of M. tuberculosisMtrB. The model is based on hydropathyanalysis using TMPRED and TOPPRED.Model I predicted 3 transmembrane (TM)helices with probability scores of 2333, 514and 2263, respectively, whereas model IIpredicted only 2 TM helices with probabilityscores of 2304 and 2263.B. Bacterial two-hybrid assay of LpqB-MtrBinteraction. Bacteriomatch Reporter E. colistrain was transformed with 9 combinations ofplasmids indicated in the table on the left.Interactions between bait (pBT-) and preypeptides (pTRG-) activated the reportergenes, which confer resistance to carbenicillin(CarbR, +, third panel from left) and produceblue colour on plates supplemented withX-Gal (fourth panel from left). Combination 2served as control as they expressed the twointeracting protein Gal11p and LGF2. Inaddition, only combination 6 and 7 expressingcytoplasmic LpqB and putative extracellulardomains of MtrB showed positive activation ofreporter genes. b-Galactosidase activity wasmeasured in reporter strains using Millerassays (right panel). Shown values are themeans of four biological replicates. Error barsrepresent standard deviations.

COOH COOH

23360 120 43 233

21513842 21563

pTRG-pBT-

Gal11LGF2

LpqB22-583

MtrB140-214

MtrB61-214

MtrB234-567

β-galactosidase (Miller Unit)0 20 40 60 80 100 120

X-GalCarbR

strain grown on carbenicillin plates (250 mg ml-1, CarbR,third panel). This result was further confirmed by theincreased production of b-galactosidase by the reporterstrain coexpressing the plasmid combinations (Fig. 6B,fourth and far right panel). The MtrB61–214 peptide displayeda weak interaction with LpqB22–583 (Fig. 6B, combination 7,right panel). Compared with the MtrB140–214 peptide thatcontains no predicted transmembrane domain, MtrB61–214

may carry one (Fig. 6A, model I, residues 120–138) thatmay localize MtrB61–214 to the membrane proximity, there-fore limiting its association with the cytoplasmic LpqBpeptide. It is also possible that MtrB61–214 not folding cor-rectly accounts for its weak interaction with LpqB. The factsthat the N-terminus of MtrB is highly positively charged (pI13) and that the second transmembrane helix in model Ihas a low probability score (Fig. 6A, legend) support thetopology in model II.Although the precise membrane topol-ogy of MtrB remains to be established experimentally,these results strongly suggest the presence of a sensordomain located within the extracytoplasmic sequence ofMtrB that interacts with LpqB.

LpqB affects phosphorylation but not expression of MtrA

To investigate whether LpqB affects expression of MtrA,two different approaches were taken. First, plasmidpVN781 expressing b-galactosidase from the mtrA pro-moter was transformed to wild-type M. smegmatis andMAR2 by electroporation. Transformants were grown on7H10-hygromycin medium and samples were collectedduring growth to determine b-galactosidase activity (seeSupporting information). There were no differences inmtrA promoter activity observed between wild-type andMAR2 (Fig. 7A, upper panel). Second, plasmid pVN762expressing c-Myc tagged MtrA from its native promoter(Table S2) was introduced to wild-type and MAR2 and theexpression was analysed by Western blot using a c-Mycantibody. Cultures of M. smegmatis strains were har-vested during growth and equal amounts of cellular pro-teins from each sample were separated on SDS-PAGE,followed by Western blot (Fig. 7B). Furthermore, to inves-tigate whether LpqB mediates MtrA expression to respondto cell wall or general antibiotic stress, mycobacterial cul-tures were exposed to vancomycin (10 mg ml-1) or eryth-romycin (100 mg ml-1) for 1 h before samples wereprepared (not shown). Expression of MtrA from its nativepromoter was not affected by LpqB in any of the condi-tions investigated. These results indicate that LpqB hasno effect on MtrA at the expression level.

To investigate whether LpqB affects the phosphoryla-tion status of MtrA, M. smegmatis strains expressingc-Myc tagged MtrA from pVN762 were labelled in vivo by[32P]-orthophosphate, followed by immuno-purification ofc-Myc tagged peptides. Purified materials were separated

by SDS-PAGE, blotted onto Whatman filter paper andexamined by autoradiography. To retain the stability of thephospho-aspartate, samples had only been treated bymild heating (see Experimental procedures) before SDS-PAGE. Under this condition, the c-Myc tagged MtrAmigrated as two forms, 25 and 50 kDa (Fig. 7C). Thehigher molecular weight form of MtrA (possibly MtrAhomodimers) was disrupted when samples were boiled(Fig. S4). More importantly, this form of MtrA was [32P]-labelled, suggesting that it was phosphorylated in vivo. Bycontrast, there was no phosphorylation signal detected forthe monomer form of MtrA (Fig. 7C). The phosphorylatedform of MtrA (MtrA-P) accounted for 38.17% of the totalMtrA signal in wild-type M. smegmatis but only 4.93% inMAR2 (Fig. 7C). This result indicates that LpqB regulatesphosphorylation levels of MtrA.

Phosphorylation status of MtrA mediates LpqB function

MtrA belongs to the OmpR/PhoB subfamily of RRs that arecharacterized by a winged helix–turn–helix DNA bindingdomain. Phosphorylation of the regulatory domain of theseregulators often correlates with induced dimerization,which enhances their ability to bind DNA and regulatetranscription (Igo et al., 1989; Fiedler and Weiss, 1995). Acrystal structure revealed that MtrA has the typicalC-terminal effector DNA binding domain as well as anN-terminal signal receiver domain with the conservedphospho-acceptor site D56 (Friedland et al., 2007). Thisaspartate residue is highly conserved and is in closeproximity to the active site of MtrA (Friedland et al., 2007).It has been established that the phosphotransfer from a HKinduces conformational changes that modulate the activi-ties of its paired RRs. To assess the in vivo functions of D56in MtrA, the mtrA gene and its D56Aor D56E mutant alleleswere cloned and overexpressed in wild-type M. smegmatisand MAR2. Similar experiments with other RRs predictedthat MtrA(D56A) would not be activated by phosphotrans-fer (Parkinson and Kofoid, 1992) and thus will remain in aninactive form. In contrast, because the structure ofglutamate mimics the phosphorylated aspartate residue,the MtrA(D56E) mutant might have a more active confor-mation, independent of the upstream signal transduction(Parkinson and Kofoid, 1992). To further avoid possiblefeedback regulation or interferences by third-partysystems, these mutated MtrA alleles were in transexpressed from the strong PSOD promoter (pVN747)instead of the native promoter. Whereas in trans expres-sion of the MtrA(D56A) allele (pVN765) in MAR2 did notlead to any detectable changes in its abnormal pheno-types, the expression in wild-type M. smegmatis resulted instriking phenotypes resembling many of those describedabove for MAR2. The M. smegmatis/pVN765 cells alsodisplayed an increased susceptibility to antibiotics,

increased cell–cell aggregation (Fig. S5), as well as signifi-cant defects in cell division control and biofilm growth(Fig. 7D). In contrast, when plasmid pVN766 expressingthe MtrA(D56E) allele from the same promoter was trans-

formed to both wild-type and MAR2, opposite effects wereobserved. MAR2 transformants expressing MtrA(D56E)displayed wild-type characteristics whereas wild-type M.smegmatis remained unchanged (Fig. 7D). Expression of

mc2155 MAR2

mc2155/pVN766mc2155 mc2155/pVN765

MAR2 MAR2/pVN765 MAR2/pVN766

IP (c-myc) Autoradiograph

MtrA monomer

MtrA dimer

Relative level

MtrA-P/MtrA

96 h72 h

nit) dnaA promoter

1000β-

Unit) mtrA promoter

96 h72 h

wild-type MtrA (pVN763) in MAR2 led to effects that aresimilar to, but less intense than, those caused by theexpression of MtrA(D56E) (Fig. S6). Together, theseresults indicate that the phosphorylation status of MtrAmediates functions of LpqB.

LpqB affects promoter activity of dnaA – an mtrAregulon gene

The only direct target of MtrA in mycobacteria thus farknown is the gene encoding the master regulator of DNAreplication DnaA (Fol et al., 2006). To further investigatewhether lpqB expression affects MtrAB function, we mea-sured the promoter activity of dnaA in the M. smegmatisstrains during growth on solid medium 7H10. Duringearlier stages of growth (48 and 72 h after inoculation),dnaA promoter activity of MAR2 was elevated comparedwith that of wild-type M. smegmatis (Fig. 7A, lower panel).These results indicate that LpqB negatively affects dnaAexpression through a control of its promoter activity.

Discussion

Computational analyses identified approximately 48 and61 putative lipoproteins encoded in the genomes of M.tuberculosis and M. smegmatis respectively (Rezwanet al., 2007). These proteins provide diverse functions inmycobacterial species, including solute binding proteinsfor efflux pumps, carriers for lipid translocation, enzymesinvolved in biosynthesis, degradation and metabolism ofcell wall constituents, as well as modulators of host celladhesion and pathogenesis of pathogenic mycobacteria(Sutcliffe and Harrington, 2004). There is growing evidencethat lipoproteins may also function in signal transductionand regulatory processes in Actinobacteria, includingMycobacterium (Steyn et al., 2003). In S. coelicolor, theextracytoplasmic lipoprotein CseA was suggested to

modulate activity of the two-component system CseB-CseC through interactions with the extracellular sensordomain of the HK CseC to respond to cell wall stress (Honget al., 2002; Hutchings et al., 2006). Analysis of Actinobac-terial genomes revealed a significant number of HKs thatare genetically linked with a putative lipoprotein, suggest-ing that such ‘three-component’ signal transductionsystems (i.e. two components plus a lipoprotein) arecommon in high G+C Gram-positive bacteria (Hoskissonand Hutchings, 2006). The putative tricistronic operonencoding MtrA–MtrB–LpqB, a member of this proposedthree-component system family, is ubiquitously present inActinobacteria (Fig. S1) but absent in other bacterialgroups, suggesting that it provides specialized functions tothese bacteria (Gao et al., 2006). Although the function ofMtrAB has been studied at some level, nothing is knownabout the lipoprotein LpqB. Here, we present evidencesupporting that LpqB functions as an accessory proteinthat modulates activity of the MtrAB system in controllinghomeostasis of the cell wall and cell division (Fig. S7).

In Corynebacterium glutamicum, both mtrA and mtrBcould be deleted whereas deletion of lpqB was lethal(Brocker and Bott, 2006), suggesting that the gene isessential in this bacterium. Likewise, a transposon sitehybridization experiment suggested that lpqB is alsoindispensable in M. tuberculosis (Sassetti et al., 2003).We show here that an lpqB transposon mutant could beisolated from the fast-growing M. smegmatis. A transpo-son insertion in lpqB abolished translation of almost two-thirds of the protein from the C-terminus (Fig. 1). Althoughsurviving the disruption, the lpqB mutant (MAR2) exhib-ited a pleiotropic phenotype that relates to defects of thecell wall and cell division. Similar to the mtrB mutant of M.avium (Cangelosi et al., 2006), MAR2 displayed a greatlyenhanced susceptibility to multiple antibiotics with adiverse array of chemical structures and mechanisms ofaction (Fig. S2, Table S1). This non-specific antibiotic

Fig. 7. Role of LpqB in expression, phosphorylation and activity of MtrA.A. Effect of LpqB on promoter activity of mtrA and dnaA. Cultures of wild-type M. smegmatis and MAR2 carrying pVN781 (upper panel) orpVN779 (lower panel) grown on 7H10 plates were collected at 48, 72 and 96 h after inoculation and total b-galactosidase activity wasmeasured using previously described methods (Timm et al., 1994). The presented data are the means of biological triplicates. Error barsrepresent standard deviations.B. Western blot analysis of MtrA expression. Wild-type M. smegmatis and MAR2 were transformed with either vector control (pVN747) or thevector expressing the c-Myc tagged MtrA from its native promoter (pVN762). Cultures were grown in a liquid medium for 48 h. Samples werecollected and treated with SDS buffer and heated for 10 min at 95°C. Total protein extracts were separated on SDS-PAGE, followed byWestern blot with c-Myc antibody to detect c-Myc tagged MtrA expression.C. LpqB affects MtrA phosphorylation states. Cultures of M. smegmatis strains expressing c-Myc tagged MtrA from pVN762 were labelled with[32P]-orthophosphoric acid. MtrA was immuno-purified using c-Myc antibody-coupled columns and separated by SDS-PAGE followed byquantitative Western blot (left panel) and autoradiography (right panel) to measure total MtrA and phosphorylated MtrA (MtrA-P) respectively.In the ‘non-boiled’ condition (37°C, 15 min followed by 60°C, 2 min), MtrA showed dimers that were labelled with 32P (left panel). Thepercentages show the ratio of dimerized MtrA (MtrA-P) versus total MtrA recovered. The presented values are the means of biologicalduplicates with variations of �2.51% and �1.4% for mc2155 and MAR2 samples respectively.D. Phosphorylation states of MtrA affect LpqB-mediated phenotypes. Two representative LpqB-related phenotypes were shown: cellmorphology and biofilm growth (see others in Supporting information). Wild-type M. smegmatis and MAR2 were transformed with vectorsexpressing the D56A (pVN765) or D56E (pVN766) alleles of MtrA from PSOD. Whereas pVN765 transformed wild-type M. smegmatis toMAR2-like, pVN766 restored wild-type-like characteristics to MAR2. Similar experiments were performed with M. smegmatis strains expressingwild-type MtrA from PSOD (Fig. S6).

sensitivity spectrum suggests that the cell wall integrity ofMAR2 is compromised.

Interrupted cell wall homeostasis often leads to collat-eral phenotypes such as alterations in surface motilityand biofilm formation (Senadheera et al., 2007). MAR2displayed an increased cell–cell aggregation in liquidmedia (Fig. 2A), which is likely the cause of the respec-tive sliding motility (Fig. 3A) and biofilm growth (Fig. 3Band C) defects. A recent study in M. tuberculosis hasrevealed a correlation of biofilm growth and the devel-opment of persisters with increased antibiotic tolerance(Ojha et al., 2008). This correlation may underlie theincreased drug tolerance observed in latent or relapsedtuberculosis. Given its role in a signal transductionpathway that is required for both antibiotic resistanceand biofilm growth of Mycobacterium, LpqB and itsassociated signal transduction network might function asa phenotypic switch contributing to the development ofdrug-tolerant persisters during biofilm growth and latentinfection of M. tuberculosis.

Cytokinetic and cell wall homeostatic controls might alsobe co-regulated by common genetic determinants. Forexample, the multiple-component signal transductionsystem YycFGHIJ in low G+C Gram-positive bacteriaco-ordinates cell division with cell wall biosynthetic pro-cesses (Bisicchia et al., 2007; Fukushima et al., 2008;Winkler and Hoch, 2008). Being a component of the septalcell division protein complex allows the HK YycG to adjustits phosphotransfer to the RR YycF in a spatial manner,thereby affecting gene expression involved in cell wallsynthesis in co-ordination with cell division processes(Fukushima et al., 2008). In support of this paradigm,MAR2 exhibited defects not only in cell wall integrity butalso cell division control. Significant increase of theaverage cell length, together with great length variation,suggests that either spatial or temporal control of celldivision was defective (Fig. 4). Again, this phenotype issimilar to the phenotypes of the mtrAB deletions. Disrup-tion of either mtrA (Möker et al., 2004) or mtrB (Brocker andBott, 2006; Cangelosi et al., 2006) leads to an increasedcell length in C. glutamicum and M. avium. Interestingly,deletion of both mtrA and mtrB in C. glutamicum increasescell length to a greater extent than either the single deletion(Brocker and Bott, 2006). Together, these results indicatethat both MtrAB and LpqB are required for the control of celllength in Mycobacterium, as well as other rod-shapedActinobacteria. Thus far, it is unclear how cell length isdetermined in this group of bacteria, which apparently lackthe cell division positioning proteins MinCD, as well as thecell shape determining MreB-like proteins (Jones et al.,2001). Future study will need to identify the nature of theupstream factors that provide the ‘cell length’ signals to theLpqB-MtrAB system, as well as the downstream regulonsresponsible for cell division.

Another intriguing feature of MAR2 is its morphologi-cal reminiscence of Streptomycetes (Fig. 4). Alteredexpression of certain cell division proteins had previ-ously been shown to switch Mycobacterium from thetypical bipolar growth to multi-polar growth and branchformation (Gomez and Bishai, 2000; Nguyen et al.,2007; Scherr and Nguyen, 2009). This morphologicalswitch possibly underlies the observed pleomorphism ofmany Mycobacterium species that might help them toadapt to environmental changes, including the nichewithin infected host cells (Chauhan et al., 2006; Scherrand Nguyen, 2009).

Whereas in vitro study of Corynebacterium suggestedthat detection of osmotic stress is mediated exclusively bythe cytoplasmic domain of MtrB (Möker et al., 2007), ourresults showed that LpqB only interacts with the extracy-toplasmic domain (Fig. 6), indicating that the latter domainmight play an important role in receiving signals fromLpqB. The discrepancy is not surprising because func-tional variations of MtrAB have been observed in differentspecies of Actinobacteria. For example, our experimentsdid not reveal a role of LpqB in culture pH control asshown for the mtrAB mutants of Corynebacterium(Fig. S8) (Möker et al., 2004). Providing cells with such adiverse array of functions would require MtrB to evolvemultiple mechanisms both for the signal reception fromvarious upstream sources, as well as for the signal deliv-ery to multiple downstream partners.

The interaction of LpqB and the sensor domain of MtrBprobably triggers autophosphorylation of the cytoplasmicdomain of MtrB, leading to the activation of MtrA anddownstream signalling cascades (Fig. S7). We show thatwhereas LpqB deletion did not affect MtrA expression(Fig. 7A and B), it altered the phosphorylation status of theprotein (Fig. 7C). This phosphotransfer deficit was likelyrooted in a failed activation of MtrB in response to unknownphysiological or environmental conditions. In support ofthis model, targeted mutagenesis experiments indicatedthat the phosphorylation status of MtrAmediates the LpqB-related characteristics (Fig. 7D and Fig. S5). Overexpres-sion of MtrA(D56A) from PSOD in wild-type M. smegmatisled to MAR2-like phenotypes (Fig. 7D) that are similar tothe phenotypes described for the mtrAB mutant of C.glutamicum (Möker et al., 2004). These results suggestthat the D56A form of MtrA was able to create a dominantnegative effect when in trans overexpressed in wild-typemycobacteria. It remains to be established how theincreased presence of the inactive form of MtrA leads tothis dominant negative effect. Given the low probability ofinactive MtrA binding DNA targets or forming higher ordercomplexes, as revealed by its crystal structure (Friedlandet al., 2007), we favour the hypothesis that MtrA(D56A)interacts with the intracellular HK transmitter domain ofMtrB, thereby outcompeting the wild-type MtrA in the phos-

photransfer reaction. Similar mechanisms of signal trans-duction delays have been reported previously with othertwo-component systems (Goymer et al., 2006). LpqB-mediated activation of MtrAB is apparently required for asuppression of dnaA expression as disruption of lpqB led toincreased dnaA promoter activities (Fig. 7A). In fact, MtrAis able to function as either an activator or a repressor ofgene transcription (Brocker and Bott, 2006). In M. tubercu-losis cells growing in macrophages, overexpression ofwild-type MtrA resulted in dramatic upregulation of dnaAexpression (Fol et al., 2006). This led the authors to con-clude that phosphorylated MtrA is an activator of dnaAtranscription (Fol et al., 2006). Contradictory to that con-clusion, simultaneous overexpression of MtrB relieves thednaA upregulation caused by the overexpression of MtrAalone (Fol et al., 2006), suggesting that the MtrB-mediatedphosphorylation of MtrA is required to suppress dnaAexpression. The crystal structure of MtrA reveals an exten-sive interface between the N-terminal phospho-receiverand the C-terminal DNA binding domain which stabilizesthe inactive conformation, and thereby may restrict thephosphorylation rate of MtrA (Friedland et al., 2007). Incertain conditions when the upstream phosphotransferrate is limited, an unmatched increased production ofwild-type MtrA (Fol et al., 2006) may lead to a reduced ratioof active/inactive forms of MtrA [similar to the overexpres-sion of MtrA(D56A)], thus lowering the MtrA-mediatedsuppression of dnaA transcription. Together with previouswork (Fol et al., 2006; Friedland et al., 2007), our resultssuggest that LpqB is an activator of the MtrAB system thatnegatively controls dnaA expression. Its analogous func-tion and mechanism of action (Fig. S7) further support thehypothesis that MtrA–MtrB–LpqB is the Actinobacterialanalogue of the YycFGHIJ system in Firmicutes (Winklerand Hoch, 2008).

The absence of two-component signal transductionsystems in animals has generated interest in their potentialas targets for future chemotherapies attacking drug-resistant infections (Okada et al., 2007; Winkler and Hoch,2008). In view of the essentiality of MtrAB in mycobacterialviability, antibiotic resistance and biofilm growth, thissystem and its accessory proteins such as LpqB may serveas promising targets for future development of new antibi-otics that ameliorate the current shortage of effective che-motherapies against multidrug-resistant and extensivelydrug-resistant tuberculosis (Nguyen and Pieters, 2009).

Experimental procedures

Bacterial strains, chemicals, media and growthconditions

All strains and plasmids used in this study and the details oftheir constructions can be found in the Supportinginformation. Wild-type M. smegmatis and its transposon-

derived mutants were grown in 7H9 liquid medium or on7H10 (Difco) or Luria-Bertani (LB) agar medium supple-mented with 0.5% Tween 80. Kanamycin was used at a finalconcentration of 50 mg ml-1. Hygromycin was used at 100 and75 mg ml-1 for Escherichia coli and mycobacteria respectively.Globomycin was a generous gift from Masatoshi Inukai(Sankyo Corporation and International University of Healthand Welfare, Japan). Preparation of competent cells andtransformation were carried out as described (Braunsteinet al., 2002). The c-Myc monoclonal antibody (9E10) wasobtained from the Developmental Studies Hybridoma Bank,University of Iowa.

Site-directed mutagenesis of D56 in MtrA

The Expand Long Template PCR kit (Roche Molecular Bio-chemicals) was used to amplify the M. smegmatis mtrAalleles from pVN762. Mutant alleles of mtrA in which thephosphorylated residue aspartate 56 (D56) was replacedby an alanine (D56A) or a glutamate residue (D56E) weregenerated by a two-stage PCR procedure. Primers MtrA2.Nand MtrA4.H were used together with either the primerpairs (mtrA-DA1 + mtrA-DA2) or (mtrA-DE1 + mtrA-DE2) toamplify the D56A or D56E allele respectively (Table S3). AllPCR products were cloned in the vector pGEM-T Easy andsequences were confirmed by sequencing. Mutant mtrAgenes were cloned into pVN747 (NdeI/HindIII), thus theirtranslation was fused to the PSOD promoter upstream(pVN765 and pVN766, Table S2).

Inhibition of signal peptidase-mediated processing ofLpqB

Mycobacterium smegmatis cultures (OD600 ~2) were split intotwo portions (20 ml) and grown for 16 h in the presence orabsence of 50 mg ml-1 globomycin. Cell lysates were pre-pared by bead beating using the Fastprep 24 (MP Biomedi-cals, Solon, OH, USA) and separated by SDS-PAGE. Tofacilitate separation of LpqB forms, samples were electro-phoresed for an extended time until proteins of molecularweights smaller than 30 kDa had run out of the gels. Isoformsof LpqB were visualized by Western blot using the anti-LpqBantibody.

Cell fractionation

Subcellular fractions of M. smegmatis cells were prepared asdescribed (Gibbons et al., 2007). Briefly, mycobacterial cul-tures (500 ml) were collected and washed with PBS (3000 g,10 min). Cell pellets were resuspended in 10 ml of PBSsupplemented with protease inhibitors and disintegrated byFrench press. Unbroken cells were removed and the total celllysate was centrifuged at 27 000 g for 30 min to pellet the cellwall. The supernatant was centrifuged at 100 000 g for 2 h toseparate the membrane fraction from the soluble fraction.

Bacterial two-hybrid protein interaction assay

Protein interaction assays were performed using the Bacte-rioMatch I reporter strain, following the manufacturer’sinstructions (Stratagene). Plasmids expressing prey and bait

peptides were constructed as described in the Supportinginformation. The strength of interaction was quantified byanalysing the b-galactosidase activity of the reporter strainsand expressed as Miller Units.

In vivo phosphorylation and immuno-precipitation

Plasmid pVN762 expressing c-Myc tagged MtrA from itsnative promoter was transformed to wild-type M. smegmatisand MAR2. Expression of c-Myc tagged MtrA was detectedby Western blot, using anti-c-Myc antibody. In vivo [32P] label-ling was performed as previously described (Radhakrishnanet al., 2008) with modifications. A single colony of cells pickedfrom a 7H10 plate was washed and grown overnight in 7H9medium to an OD600 of 0.5. This culture was used to inoculate50 ml 7H9 medium and grown to an OD600 of 0.8, washed andresuspended in 1 ml phosphate-depleted Sauton’s medium.The culture was then labelled for 1 h at 37°C using 100 mCi of[g-32P]-orthophosphoric acid (Perkin Elmer). Following celllysis by bead beating, proteins were immuno-precipitatedwith the ProFound c-Myc Tag IP/CoIP Kit according to themanufacturer’s instructions (Pierce, Rockford, IL, USA). Thebound materials were eluted from the columns followed by amild heating (37°C 15 min followed by 60°C 2 min). Sampleswere resolved by SDS-PAGE, gel dried onto Whatman Paper,and [32P]-labelled MtrA was quantified using a Storm 820PhosphorImager and ImageQuant software version 4.0(Molecular Dynamics) and normalized to the relative MtrAcontent as determined by immunoblotting of the sameimmuno-precipitated materials.

Antibody production

Anti-LpqB polyclonal antibody was produced in rabbits byusing a synthetic 15-amino-acid peptide sequence identicalin M. smegmatis and M. tuberculosis LpqB proteins (MDPD-VLLREFLKATA) (Affinity BioReagents, Golden, CO, USA).The antibody was purified by affinity chromatography usingthe synthetic peptide as a ligand.

Other methods

Details of other procedures can be found in the Supportinginformation.

Acknowledgements

We thank Michael Niederweis, Masatoshi Inukai, ElliottCrooke, David Alland, Sabine Ehrt and Dirk Schnappinger forproviding materials, Bing Liu, Daniel Kiss and MeganMamolen for technical assistance, Piet de Boer, Kien Nguyenand Abram Stavitsky for critical reading of the manuscript.This work was supported by start-up funds from the School ofMedicine, a STERIS Infectious Diseases Research Awardand CFAR Developmental Awards from the Case/UH Centerfor AIDS Research (AI36219) to L.N. S.O is a trainee of theFogarty AIDS International Training and Research Program(AITRP) at Case School of Medicine.

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A lipoprotein modulates activity of the MtrAB two-component system to provide intrinsic multidrug...

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