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Bacterial outer membrane channel for divalent metalion acquisitionThomas H. Hohlea, William L. Franckb, Gary Staceyb, and Mark R. O’Briana,1

aDepartment of Biochemistry, State University of New York, Buffalo, NY 14214; and bNational Center for Soybean Biotechnology, Division of Plant Sciences,University of Missouri, Columbia, MO 65211

Edited by Eva Kondorosi, Institute for Plant Genomics, Human Biotechnology and Bioenergy, Szeged, Hungary, and approved August 9, 2011 (received forreview June 22, 2011)

The prevailing model of bacterial membrane function predicts that

the outer membrane is permeable to most small solutes because ofpores with limited selectivity based primarily on size. Here, we

identified mnoP  in the Gram-negative bacterium Bradyrhizobium

 japonicum as a gene coregulated with the inner membrane Mn2+

transporter gene mntH . MnoP is an outer membrane protein ex-

pressed specifically under manganese limitation. MnoP acts as

a channel to facilitate the tranlocation of Mn2+, but not Co2+ orCu2+, into reconstituted proteoliposomes. An mnoP mutant is de-

fective in high-affinity Mn2+ transport into cells and has a severe

growth phenotype under manganese limitation. We suggest that

the outer membrane is a barrier to divalent metal ions that requires

a selective channel to meet the nutritional needs of the cell.

metalloregulation | regulation

Membranes control the traf ficking of solutes into cells andorganelles, and thus play an essential role in maintaining

homeostasis. Mitochondria, chloroplasts, and Gram-negativebacteria have both an outer and inner membrane in which sol-utes must traverse. Inner membrane permeability is highly se-lective because of transporters with high specificity for ligands.Conversely, the outer membrane is generally assumed to bemuch less discriminating toward small hydrophilic solutes be-cause of channels with no or broad substrate specificity thatpermit diffusion from the environment into the intermembrane

space (1–

6). These channels are transmembrane proteins com-posed of amphipathic β-strands that form a β-barrel pore thatallows solute translocation across the outer membrane. Somechannels, called porins, discriminate by size and behave as amolecular sieve with a limit of about 600 Da. Other channelssimilar to porins bind solutes with low af finity, and thus are moreselective but have relativity broad substrate specificities (7, 8).Growth phenotypes of bacterial mutants defective in genesencoding low-selectivity channels have not been demonstrated inmost cases. Thus, it is dif ficult to confirm that a given channelprevents nutrient limitation to the cell.

Metals are required for many cellular processes and must beacquired from the environment. Nutritional metals, such asmanganese, copper, cobalt, and zinc are available as the divalent

cation in aerobic environments, and are thus soluble. Whereascytoplasmic (inner) membrane transporters of free metal ionsare well characterized in bacteria (9–12), translocation across theouter membrane and into the periplasm has not been described.In principle, outer membrane pores with no or low selectivity should readily accommodate the diffusion of these small, solublenutrients that are needed only in low quantities (4, 5). However,bacteria occupy niches in which the metal is often scarce, andcells can readily take up metals available in the low nanomolarrange (13). Thus, we questioned whether simple diffusion acrossthe outer membrane down such a shallow gradient via a non-selective pore is suf ficient to satisfy the nutritional needs of the cell.

The Gram-negative bacterium Bradyrhizobium japonicum isa model organism for studying metal metabolism and regulation

in the α-Proteobacteria, a large and diverse taxonomic group thatincludes pathogens, symbionts, and bacteria that occupy many other ecological niches (14). B. japonicum maintains a constantintracellular manganese level over a wide range of manganese inthe growth medium because of a regulated divalent manganese(Mn2+) transport activity (13). MntH is a high-af finity inner-membrane Mn2+ transporter that is specifically expressed undermanganese limitation, allowing the uptake of extracellular Mn2+

in the nanomolar range (13). The mntH  gene is transcriptionally regulated by the Mur repressor (previously referred to as Fur inthis bacterium). Association of Mn2+  with Mur confers DNA binding activity on the protein, resulting in occupancy of the mntH  promoter by Mur and transcriptional repression (13, 15).The responsiveness of the Fur-like regulator Mur to Mn2+ in B. japonicum and other α-Proteobacteria rather than to Fe2+ (13,15–19) may reflect an adaptation by this group to a more man-ganese-dependent metabolism. In support of this, an mntH mutant of  B. japonicum has a severe growth phenotype (13),

 whereas other bacterial mntH  mutants do not unless they areexposed to oxidative stress (20). Manganese also controls ironhomeostasis in B. japonicum by affecting stability of the tran-scriptional regulator Irr (21, 22).

In this study, we identified a regulated and selective outermembrane channel required for Mn2+ acquisition and growthunder metal limitation in B. japonicum.

ResultsIdentification of a Candidate Outer Membrane Protein-Encoding Gene

That Is Coregulated withmntH . To search for new genes involved inMn2+ acquisition, we used whole-genome microarray analysis tosearch for those that are coregulated with mntH , the geneencoding the inner-membrane Mn2+ transporter MntH. The mntH  gene is repressed by Mur in the presence of manganeseand derepressed under manganese limitation (13, 15). Thus,using wild-type cells grown in manganese-replete medium as thereference, we screened for genes that were up-regulated both inthe wild-type grown under manganese limitation and in a mur strain grown in manganese-replete medium. Only 12 genes ful-filled both criteria (Fig. S1). Among them were mntH and irr , thelatter of which was shown previously to be controlled by man-ganese and Mur (15, 23).

The blr0095 gene, annotated as a hypothetical protein, showedthe greatest response to manganese and Mur in the microarray analysis. This gene was renamed mnoP  (Mn-responsive outer

Author contributions: T.H.H., G.S., and M.R.O. designed research; T.H.H. and W.L.F. per-

formed research; T.H.H., W.L.F., G.S., and M.R.O. analyzed data; and T.H.H. and M.R.O.

wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequence reported in this paper has been deposited in the Gene

Expression Omnibus database (accession no. GSE30932).

1To whom correspondence should be addressed. E-mail: mrobrian@buffalo.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1110137108/-/DCSupplemental.

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membrane protein) for reasons described below. The mnoP geneis not part of an operon because it has its own transcription startsite (see below), and the gene downstream of it is in the oppositeorientation. MnoP is predicted to contain extensive β-sheetsecondary structure, the signature of transmembrane outermembrane channels, and thus it was studied further. To confirmthe microarray data, we examined the control of  mnoP  usingquantitative real-time PCR (qPCR) and compared the expres-sion profile to that of mntH (Fig. 1 A and B). The mnoP gene was

strongly induced under low manganese growth conditions, andrepression observed in the presence of manganese in the parentstrain was relieved in a mur mutant. Thus, mnoP  and mntH  areregulated similarly. Moreover, mnoP  mRNA was not affected by the iron status in the growth medium (Fig. 1 A), showing that highexpression under manganese limitation was not the result of a general stress response. In addition, some iron-responsivegenes are aberrantly expressed in a B. japonicum mur  mutant(24, 25), but mnoP  was not.

Mur Binds the mnoP  Promoter in Vivo and in Vitro in a Manganese-

Responsive Manner. The mntH gene is controlled by direct bindingof Mur to its promoter in the presence of manganese (13). Muroccupancy of the mnoP promoter was examined by cross-linking/ 

immunoprecipitation (IP). DNA that coprecipitated with anti-Mur antibodies was analyzed by qPCR using primers that amplify the promoter region. The promoter regions of  mnoP  (Fig. 1C)and mnoP  (Fig. 1 D) were occupied by Mur in the parent strainonly when manganese was present in the growth media. Muroccupancy was independent of the iron status. As an additionalcontrol, cross-linking/IP was also carried out in a mur  mutant(Fig. 1 C and D). Hence, Mur occupancy of the mnoP  promotercorresponds with repression in the presence of manganese, as

 was observed previously for mntH .To identify the Mur binding site on the mnoP  promoter, we

determined the transcriptional start site using 59 RACE and thenanalyzed the region upstream of it (Fig. 2). DNase I footprintinganalysis showed that recombinant Mur bound the mnoP promoteronly in the presence of Mn2+, with maximum occupancy in the

−47 to

−13 region relative to the transcription start site (Fig. 2 A

and B). Examination of theprotected region of DNA on the mnoP promoter revealed that the sequence contains three imperfectdirect hexameric repeats (Fig. 2 B), similar to those observed

 within the mntH (13) and irr (23) promoters (Fig. 2C). Collectively,the data show that mnoP  and mntH  are coregulated by directbinding of Mur to similar cis-acting elements on the respectivepromoters.

MnoP Is an Outer Membrane Protein and Predicted to Form a β-Barrel

Channel. Bioinformatic analysis (26) of MnoP shows it to behomologous to two families of outer membrane transmembraneβ-barrel proteins. The N-terminal region is similar to the surface

antigen-2 superfamily, and the C-terminal portion is predicted tobelong to the OprB superfamily. Pseudomonas aeruginosa OprBis a carbohydrate channel (8), but other members of the OprBsuperfamily have not been characterized. MnoP was modeledusing the HHpred server (27) and predicted to contain 14β-sheets organized as a β-barrel protein (Fig. 3 A). The β-barrelcomprises 580 amino acids of the protein, and the remaining C-terminal 70 amino acids were unmodeled.

Genes encoding MntH and MnoP tagged with c-Myc and HA,respectively, at the C-termini and under the control of theirendogenous promoters were constructed and introduced into B. japonicum cells. Inner- and outer membrane fractions wereprepared from cells grown in manganese-replete or manganese-limited conditions, and analyzed by Western blotting using

antibodies directed against the respective tag (Fig. 3 B). MnoP was found in the outer membrane fraction, in agreement with itspredicted structure. MntH was confirmed to be an inner-mem-brane protein. Moreover, both MntH and MnoP were detectedonly in samples from cells grown under manganese limitation,

 which correlates with induction of the mRNAs that encode them.MntHappearedasadoubletof52and55kDaonSDS/PAGE,in

reasonable agreementwith thepredictedsizeof 50 kDa. Thelarger(minor) band may be an unprocessed peptide or a modified vari-ant. MnoP migrated at about 52 kDa, which differed substantially from the calculated mass of 69 kDa. This aberrant migration iscommon for outer membrane β-barrel proteins (e.g., ref. 28).

MnoP Protein Facilitates Mn2+ Entry in Reconstituted Liposomes. Theouter membrane location of MnoP and its putative structure pre-

dict that it is a channel protein. To test this theory, recombinantMnoP protein was overexpressed, purified, and incorporated intoliposomes, forming proteoliposomes (see Materials and Methods).Transport activity was monitored using a swelling assay (29) in

Fig. 1. Coregulation of mnoP  and mntH  in a manga-

nese- and Mur-responsive manner. ( A and B) Analysis of

mnoP  and mntH  mRNA by qPCR in wild-type or mur 

mutant cells grown in media supplemented with or

without 50 μM MnCl2 and 20 μM FeCl3. The data are

expressed as the relative starting quantity (SQ) of mnoP 

or mntH  mRNA normalized to the housekeeping gene

gapA, and presented as the average of triplicate sam-

ples ± the SD. (C  and D) In vivo occupancy of the mnoP 

and mntH promoters by Mur in cells grown as described

above by cross-linking followed by IP using anti-Mur

antibodies. The precipitated DNA was analyzed by qPCR

using primers that amplify promoter DNA from mnoP 

or mntH . The data are expressed as the relative starting

quantity of immunoprecipitated DNA normalized to

a mock pull-down without the primary antibody.

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  which uptake of a substrate causes the liposome to swell tomaintain osmotic balance. Swelling results in a decrease in theoptical density of the liposome suspension. In the absence of 

MnoP, the liposomes were impermeable to any of the metal saltstested, as seen by the lack of swelling (Fig. 4 A). However, pro-

teoliposomes containing MnoP swelled in the presence of MnCl2or MnSO4 (Fig. 4 B). Divalent cobalt and copper (Co2+ and Cu2+)are stable in aerobic environments, and therefore CoSO4 and

CuSO4 were also tested in the liposome assay (Fig. 4 B). Swelling was not observed with either metal salt. These observations showthat MnoP confers permeability to Mn2+ and is a selectivechannel.

MnoP Is Essential for High-Affinity Mn2+ Transport Activity in Cells.

Previously, we reported that B. japonicum has a high-af finity Mn2+

transport activity that is induced under manganese limitation (13).To determine whether MnoP is required for this activity, we mea-sured uptake using 5 nM 54Mn2+ as the initial concentration in an mnoP  mutant grown in high- or low-manganese media, and com-pared them to the wild-type and mntH strains (Fig. 5). Mn2+ trans-port activity was very low in all strains grown in high-manganesemedia(Fig.5 A), conditions where mntH and mnoP arenot expressed(Figs. 1 A and B,and3 B).Mn2+ transport was induced in the parentstrain grown under manganese limitation, but this activity was se-

 verely diminished in the mnoP mutant, as well as in the mntH strain(Fig. 5 B). Thus, MnoP is required for high-af finity Mn2+ uptake.

To quantify the contribution of MnoP to cellular Mn2+

transport, we measured the initial velocity of Mn2+ uptake asa function of the Mn2+ concentration in the parent and mutantstrains, and determined the apparent K m values using a Line-

  weaver-Burk plot analysis (Fig. 5C). The apparent K m for Mn2+

uptake by the parent strain was 87 nM (Fig. 5 C). In the mnoP mutant, the apparent K m was 1,656 nM, 19-fold higher than forthe parent strain. Thus, MnoP contributes substantially to high-af finity Mn2+ uptake by cells. The Lineweaver-Burk plot for the mntH strain yielded a positive abscissa value (Fig. 5C); thus, theapparent K m value could not be determined, presumably becauseof the near absolute requirement of MntH for Mn2+ uptake.

Fig. 2. Identification of the Mur binding site on the mnoP 

promoter in vitro by DNase I footprinting. ( A) Protection of

DNA from DNase I digestion by Mur was carried out in the

presence or absence of MnCl2 using 0, 2, 5, 10, 25, or 50 nM

Mur. The DNA was radio-labeled at the 59 end of the non-

template strand with respect to the mnoP  gene, and thus the

39 end is at the top of the gel. The arrows denote the sites of

the three hexamer repeats. (B) The sequence of the protectedregion of the mnoP  promoter are shown as lowercase letters.

The bent arrow represents the transcription start site. The di-

rect repeats are shown by the straight arrows. The underlined

sequence shows the putative −35 region of the promoter. (C )

A consensus binding site for B. japonicum Mur identified using

the Mur-binding sites of mntH , mnoP , and irr as determined by

the MEME algorithm. The Mur binding regions on the mntH 

and irr promoters were determined previously, as described in

the text.

Fig. 3. Localization of MnoP to the outer membrane. ( A) Predicted model

of MnoP β-barrel structure. (B) Western blots analysis of c-myc–tagged MnoP

or HA-tagged MntH in the outer membrane (OM) or inner membrane (IM)

fractions of cells grown in the presence (+) or absence (−) of 50 μM MnCl2.

Thirty micrograms of protein was loaded onto each lane.

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MnoP Is Required for Growth Under Manganese Deficiency. We wanted to address whether the defect in Mn2+ transport in the mnoP mutant limits cell growth under manganese limitation. Todo this, we examined the growth of the parent, mntH , and mnoP strains on solid media containing 25 μM EDDHA (ethylenedi-amine-di- o-hydroxyphenylacetic acid) as a metal chelator (Fig. 6).Heme was used as the iron source because heme iron is not che-

lated by EDDHA. The parent strain grew without manganesesupplementation (Fig. 6), indicating that the cells were able toscavenge it from the medium. However, neither the mnoP  nor mntH mutants grew under these conditions, showing that MnoPand MntH are necessary for growth under metal limitation. Thegrowth phenotype of the mnoP  strain was partially restored withsupplementation with 5 pmol MnCl2 to a paper filter in the centerof the plate, as observed by the zone growth around the filter (Fig.6). No growth was observed when the filters were spotted withdivalent salts of zinc, copper, cobalt, or nickel instead of manga-nese. The mntH strain had a more severe phenotype, requiring 50pmol MnCl2 to partially restore growth (Fig. 6). These data showthat MnoPis required for cell growth under manganese limitation.

Discussion

The prevailing model of outer membrane structure and functionpredicts that small soluble substrates, such as divalent metalcations, should traverse it via channels with no or low selectivity (4, 5). Nevertheless, the present study shows that manganeseacquisition requires the regulated expression of a selective outermembrane channel to translocate the metal from the environ-ment into the periplasm. Moreover, the absence of this channelrenders the outer membrane a diffusion barrier that prevents thecell from meeting its nutritional manganese requirement. Thisconclusion is based upon several observations. First, mnoP  ex-pression is not constitutive but is specifically expressed undermanganese limitation, and is coordinated with the inner mem-brane transporter gene mntH . Both gene promoters contain aMur-binding site that permits occupancy in the presence of 

manganese and derepression when the metal is scarce. Second,MnoP allows the translocation of Mn2+ into proteoliposomes,but Co2+ or Cu2+ are not transported. The atomic radii of Mn2+,

Fig. 4. MnoP-mediated transport of divalent metals in proteoliposomes.Liposome swelling assays were initiated by the addition of MnCl2 (•), MnSO4

(B), CuSO4 (▲), CoSO4 (;), or metal-free water (■) to a suspension con-

taining 1 mg/mL reconstituted liposome, and either ( A) 0 or (B) 1 μg/mL

recombinant MnoP. The suspension was monitored at an optical density of

400 nm (OD400) for 90 s.

Fig. 5. Mn2+ uptake by B. japonicum parent and mutants strains. Cells of

the parent strain (■), the mntH  strain (•), or the mnoP  strain (◆) were

grown in ( A) high- or (B) low-manganese media. At time 0, 5 nM 54Mn was

added to the assay medium and aliquots were subsequently taken at various

time points and counted. (C ) Lineweaver Burk plot. 54Mn2+ uptake was

measured for the parent strain (■), mntH  (•), or mnoP  (◆) for 5 min at

concentrations of Mn2+ ranging from 75 to 250 nM.

Fig. 6. Dependence on manganese supplementation for growth of the B.

 japonicum parent strain and mntH and mnoP mutants: 106 cells were plated

on solid low-metal media supplemented with 1 μM heme as an iron source

and 25 μM EDDHA as a metal chelator. Next, 0, 5, or 50 pmol MnCl 2 was

spotted onto a 6-mm filter disk located in the center of the plate. Growth

around the filter disk was assessed visually. Samples were performed in

triplicate, and one of each is shown in the figure.

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Co2+, and Cu2+ are very similar to each other, as are the re-spective hydrated radii (30). Thus, the selectivity of MnoP is very unlikely to be based on size or valency alone. Third, MnoP wasrequired for transport of Mn2+ into cells in the low nanomolarrange of metal and increased the apparent af finity of the metalfor cells by almost 20-fold. Finally, MnoP was necessary to sup-port bacterial growth under manganese limitation, and under-scores the physiological relevance of channel.

In a few bacteria, Ni2+ (31) and perhaps Zn2+ (32) can be

taken up across he outer membrane as metal chelates involvingan energy-dependent receptor and auxiliary proteins, similar to

 what is observed for ferric (Fe3+) iron. In Helicobacter pylori, thechelated nickel-uptake system functions specifically at low pH(31), but the mechanism of free Ni2+ uptake across the outermembrane is unknown.

The outer membrane of  P. aeruginosa is moreselective than thatof  Escherichia coli, and outer membrane channels with some se-lectivity have been described (3, 4). The OprB carbohydratechannel is induced by the presence of substrate in the millimolarrange and transports a wide range of structurally diverse carbonsources, such as glycerol, glucose, and trisaccharides (8). In con-trast, mnoP derepression requires substrate limitation, and MnoPcan discriminate between similar ions. The phosphate channel

OprP is produced in response to phosphate limitation and trans-ports numerous anions, but the induction threshold is 150 μMsubstrate (33), which is much higher than the manganese con-centration needed for mnoP  expression. Although nutritionalmetals are needed in small quantities compared with carbohy-drates or phosphate, they are often available in low amounts, andthe present work indicates that simple diffusion across the outermembrane cannot support cellular needs. From this indication, itis likely that acquisition of other soluble metal nutrients requiresselective channels in bacteria. Similarly, mitochondria and chlor-oplasts are metal-rich organelles and contain numerous β-barrelouter membrane proteins, most of whose functions are not known(1). Similarity searches did not find obvious MnoP homologs, butfurther studies intounderstanding the basis of selectivity may yieldnew criteria on which to base a more refined search.

Materials and MethodsStrains and Media. B. japonicum strain USDA110 is the parent strain used in

this study. Strains GEM4 (34), mntH ΩΔ (13), and mnoP ΩΔ (present study) are

mutant derivatives of the parent strain containing a DNA cassette encoding

spectinomycin and streptomycin replacing the mur , mntH , and mnoP genes,

respectively. B. japonicum strains were routinely grown at 29 °C in glycerol-

salts yeast (GSY) medium as previously described (35). For low manganese

and iron conditions, modified GSY was used, containing 0.5 g$L−1 yeast

extract instead of 1 g$L−1, with no exogenous manganese or iron added. The

actual concentrations of manganese and iron in unsupplemented media are

0.2 μM and 0.3 μM, respectively, as determined by atomic absorption using

a Perkin-Elmer model 1100B atomic absortion spectrometer. High-metal

media was supplemented with either 50 μM MnCl2, 20 μM FeCl3, or both.

Microarray Analysis. Two comparisons were carried out: wild-type cells grown

in manganese-limited media vs. manganese-replete conditions, and the mur 

mutant vs. the wild-type grown under manganese-replete conditions. Three

biological replicates of each strain and growth condition were analyzed.

RNA was isolated from B. japonicum cells using a hot phenol-extraction

method and cDNA synthesized as previously described (36). Microarray

analysis was carried out using oligonucleotides printed on Corning epoxy-

coated slides as described previously (37). The slides were read at the

Microarray and Genomics Core Facility at the Roswell Park Cancer Institute

(Buffalo, NY). The microarray data have been deposited in the Gene Ex-

pression Omnibus (GEO), and are accessible through GEO Series accession

number GSE30932. Gene names and annotations are from the published

genome database (38) (http://genome.kazusa.or.jp/rhizobase/ ).

Analysis of RNA by qPCR. Expression levels of selected genes were determined

by qPCR with iQ SYBR green supermix (Bio-Rad) using iCycler thermal cycler

(Bio-Rad). RNA was isolated from B. japonicum cells using a hot phenol-ex-

traction method as previously described (36). cDNA was synthesized from 5

μg total RNA using iScript cDNA synthesis kit (Bio-Rad). qPCR reactions were

carried out as previously described (13). Data are expressed as average of

three triplicates and the SD is represented by the error bars.

Quantitative in Vivo Cross-Linking and IP. For quantitative in vivo cross-linking

and IP, 200-mL cultures of parent strain USDA110 or mur  strain GEM4 were

grown under low- or high-manganese and low- or high-iron conditions to

midlog phase (OD540 0.4–0.6). Cross-linking and IP were performed as de-

scribed previously (39). Immunoprecipitated DNA of selected promoter

regions was quantified using qPCR as described above, using 1-μL coimmu-noprecipitated DNA template. The data were normalized to mock pull-down

sample in which primary antibody was excluded from the reaction.

DNase I Footprinting Analysis. DNase I footprinting analysis examining mnoP 

promoter DNA protected by Mur in the presence or absence of 100 μM

MnCl2 was carried out as described previously (13). The reactions were car-

ried out in the presence 0 to 50 nM Mur.

Cellular Location of MnoP and MntH. MnoP-c-myc–tagged protein was

expressed using a very low-copy plasmid (pVK102) and transcribed from the

mnoP  promoter in the parent strain. A gene encoding MnoP-HA–tagged

protein run off of the mntH  promoter region was integrated into the ge-

nome of the parent strain. Next, 100 mL of cells harboring these constructs

grown to midlog phase were harvested by centrifugation and washed twice

in PBS (PBS). Cells were resuspended in PBS containing 2 mg/mL lysozyme.

Cell suspensions were run through the French press twice before beingsubjected to ultracentrifugation at 185,000 × g (Ti70.1 rotor, Beckman) for

90 min. The supernatant containing the soluble fraction was discarded. The

pellet was resuspended in PBS containing 2% sarkosyl by rotating on a slight

angle for 2 h at room temperature. The suspension was subjected to ultra-

centrifugation as described above, and the supernatant containing the in-

ner-membrane fraction was saved at −20 °C. The pellet was resuspended in

PBS containing 0.5% SB3-14 (Sigma) detergent by rotating on a slight angle

at room temperature overnight. The ultracentrifugation step was repeated,

and the supernatant containing the outer membrane fraction was saved at

−20 °C. Proteins were quantified using the Bradford reagent (Bio-Rad) and

30 μg were run on a 10% SDS/PAGE. Western blots were used to visualize

MnoP-c-myc and MntH-HA using α-c-Myc and α-HA antibodies (Sigma), re-

spectively, at 1:1,000 titer, incubating for 1 h. Bands were visualized using

horseradish peroxidase-conjugated goat anti-rabbit IgG and developed us-

ing the Western Lightning ECL reagent (PerkinElmer).

Protein Overexpression and Purification. The mnoP genewas amplifiedbyPCR

and cloned into pETite C-His vector (Lucigen) containing a C-terminal 6xHis

tag. The vector with insert was transformed into chemical competent BL21

(DE3) E. coli cells. Cells were inoculated from an overnight culture grown in

Luria-Bertani media containing 20 μg/mL kanamycin into 1 L of fresh 2×YT

mediumcontaining 2μg/mLkanamycin.Overexpressionwas inducedin cells at

the midlog phase by the addition of 0.5 mM isopropyl-1-thio-β-D-galactopyr-

anoside (IPTG) at 37 °C for 4 h with shaking. Cells were pelleted by centrifu-

gation, washed in phosphate-binding buffer (50 mM NaH2PO4 and 300 mM

NaCl, pH 8.0), and subsequently resuspended in 15 mL phosphate-binding

buffercontaining 8 M urea. Cells were disruptedby passage through a French

pressure cell at 1,200 psi and clarified by centrifugation at 13,000 × g for 10

min. Two milliliters of Ni-NTA slurry (Qiagen) was washed in phosphate

binding buffer containing 8 M urea before being added to the cleared lysate

and rocked for 60 min at room temperature (22 °C).

The Ni-NTA slurry-protein mixture was poured into a column and washed

four times with phosphate wash buffer (50 mM NaPO4, 300 mM NaCl, 8 M

Urea, and 20 mM imidazole, pH 8.0) and once with phosphate wash buffer

containing 10% glycerol. His-MnoP was eluted using phosphate elution

buffer (50 mM NaPO4, 300 mM NaCl, 8 M Urea, and 250 mM Imidazole, pH

8.0). The purified protein was run through an FPLC buffer exchange column

so that the final buffer consisted of 0.1 mM Tris-HCl, pH 8.0, and 8 M Urea.

Liposome Swelling Assay. To make liposomes, 100 mg of 1,2-dilauroyl- sn-

glycero-3-phosphocholine (Avanti Polar Lipids) were dissolved in 1 mM

chloroform in a 50-mL glass round-bottom flask. The flask was flushed with

nitrogen gas until the chloroform was evaporated and a lipid film remained.

The lipids were hydrated in 1 mL 0.1 mM Tris-HCl, pH 8.0, vortexed until

lipids were in solution, and subjected to five freeze-thaw cycles using liquid

nitrogen and a 37 °C water bath. Next, 400 μg recombinant unfolded MnoP

was diluted about 12-fold in the lipid suspension to promote folding. The

protein-liposome suspension was then extruded 16 times through two 200-

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nM pore size polycarbonate filters (Whatman). The protein-liposome sus-

pension was transferred to a 6-mL glass culture tube and allowed to sit at

room temperature for 2 h before the swelling assay. The liposome without

protein was prepared the same way as described, but instead of protein, 0.1

mM Tris-HCl, 8 M urea, pH 8.0 was added to the suspension. Liposome

swelling assays (29) were performed to monitor MnoP specific transport of

various substrates. The assay was initiated by the addition of 1 mM substrate

or metal-free water to a solution of 0.1mM Tris-HCl,6.7 mM urea, 1 mg$mL−1

reconstituted liposome, and either 0 or 1 μg$mL−1 MnoP. The suspension was

monitored at an optical density of 400 nm (OD400) every 10 s for 90 s. Proteins

successfully folding into liposomes were estimated by pelleting 50 μL pro-teoliposome solution at 14,000 × g for 5 min, followed by washing and

resuspending the proteoliposome in 0.1 mM Tris-HCl, pH 8.0, and the protein

concentration determined using the Bradford method (Bio-Rad).

54Mn Uptake Assay. The ability of the B. japonicum parent strain and mutants

to transport 54Mn2+ was determined, as previously described (13). For de-

termination of initial velocities, 54Mn2+ uptake was measured for 5 min

within the linear range for uptake. The data were analyzed by Lineweaver-

Burk plots and the apparent K M and V max values reported were derived from

these analyses.

Growth Disk Assays. For growth disk assays, 106 cells of the parent, mntH  ΩΔ,

or mnoP  ΩΔ strain were quickly vortexed into soft-agar medium containing 1

μM heme as an iron source and 25 μM EDDHA as a metal chelator at 50 °C

and then poured onto plates. After solidification of the agar medium,

Whatman paper disks (6-mm diameter) were placed at the center of the

plate and spotted with 5 μL of 10 μM or 1 μM MnCl2 or metal-free water asa negative control. Growth around the disk was assessed visually.

ACKNOWLEDGMENTS. This work was supported by National Institutes ofHealth Grant R01 GM067966 (to M.R.O.), National Science Foundation GrantIOS-1025752 (to G.S.), and National Institutes of Health Training Grant T32AI707614 (to T.H.H.).

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Supporting Information

Hohle et al. 10.1073/pnas.1110137108

Fig. S1. Identification of genes responsive to manganese and Mur. Genes repressed by manganese and derepressed in a mur  mutant were identified by

whole-genome microarray analysis. ( A) Graphical representation and overlap of genes that are up-regulated in the parent strain in response to manganese

limitation (290) and genes that are up-regulated in a mur  background compared with the parent strain grown in high-manganese media (207). (B) Twelve

genes found in both microarray analyses are listed.

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