Functional characterization of RhuB as a second TonB2-dependent hemin receptor in Riemerella anatipestifer CH-1

ABSTRACT In the previous study, it was shown that Riemerella anatipestifer (R. anatipestifer, RA), a pathogen in ducks and some other birds, encodes a hemin uptake system. The R. anatipestifer hemin uptake receptor RhuR is a TonB2-dependent hemin transporter. However, it remains unclear whether R. anatipestifer encodes additional TonB-dependent hemin transporters. Herein, we demonstrated that R. anatipestifer hemin uptake receptor B (RhuB) of R. anatipestifer CH-1 (RA CH-1) was negatively regulated by iron and mediated by the Fur protein, and knocking out rhuB damaged the ability of RA CH-1 to utilize iron from duck hemoglobin (Hb) but not that from duck serum. Moreover, the ability to use iron from Hb was restored by the expression rhuB in trans. Furthermore, the RhuB of RA CH-1 is a membrane protein, and recombinant RhuB could bind hemin at a 1:1 molar ratio in vitro. Compared to that of ΔtonB1ΔrhuR, the ability of ΔtonB1ΔrhuRΔrhuB to utilize hemin was impaired; meanwhile, compared to that of ΔtonB2ΔrhuR, the hemin utilization ability of ΔtonB2ΔrhuRΔrhuB was not affected, indicating that RhuB is a TonB2-dependent receptor. Compared to ΔrhuB, ΔrhuBΔrhuA did not affect hemin utilization. However, compared to ΔrhuA, ΔrhuBΔrhuA had reduced ability to utilize hemin, suggesting that RhuA relies on RhuB for its activity. Finally, the deletion of rhuB did not affect the virulence of RA CH-1. These results suggested that RhuB encodes a TonB2-dependent hemin receptor. The characterization of the second TonB-dependent receptor in R. anatipestifer enriches our understanding of the hemin uptake system of this bacterium. IMPORTANCE Iron is essential for the survival of most bacteria, and hemin of hemoglobin can serve as an important iron source. In our previous studies, we showed that R. anatipestifer CH-1 encodes a TonB2-dependent hemin receptor RhuR, which is involved in hemin uptake. The deletion of rhuR did not abolish hemin utilization by RA CH-1. We hypothesized that additional hemin uptake systems exist in this bacterium. In this study, we identified the second TonB2-dependent hemin receptor RhuB in RA CH-1 through hemin utilization, protein localization, and hemin-binding experiments. The duck infection model showed that the deletion of rhuB did not affect the virulence of RA CH-1. This study is not only important for further understanding the hemin utilization mechanism of R. anatipestifer, but also for enriching the hemin uptake transporters of gram-negative bacteria.

To overcome this obstacle, some bacteria secrete siderophores to mobilize iron from their hydroxide polymers or host iron-binding proteins, such as transferrin (2).Fe-bound siderophores are transported across the bacterial outer membrane by specific TonBdependent transporters, whereas ATP-binding cassette (ABC) transporters are responsi ble for the transportation of Fe-bound siderophores through the inner membrane.In the cytoplasm, iron is released from the siderophores by siderophore-interacting proteins (5).For hemin utilization, gram-negative bacteria encode specific TonB-dependent hemin receptor(s) for transport, which are degraded by hemin oxygenase or hemin-degrading proteins to release iron (6).To maintain iron homeostasis in cells, genes encoding iron or hemin uptake systems in most gram-negative bacteria are transcriptionally regulated by iron in a pathway mediated by the Fur protein (4,7).When iron levels are too high in the cytoplasm, iron-bound Fur binds to the promoter region of iron or hemin transporter genes to repress gene transcription.Conversely, lower external iron can cause the dissociation of Fur from the promoter region of iron or hemin transporter genes to promote gene transcription (4).Because free iron and hemin are almost nonexistent in the host, these uptake systems are important for bacterial pathogens during infection.
R. anatipestifer genome encodes at least 31 putative TonB-dependent receptors (TbdRs) (21); however, the physiological roles of most of these putative TbdRs remain largely unknown.In our previous study, we showed that the outer membrane heminbinding protein RhuA participates in hemin transport through the TonB2-dependent hemin receptor RhuR in R. anatipestifer CH-1 (22).In addition to RhuR, other TonBdependent receptors, such as B739_1068 in RA CH-1, are upregulated under iron-limited conditions (23).In this study, we aimed to show that the B739_1068 of RA CH-1 is a TonB2-dependent membrane protein, and B739_1068 could utilize hemin from duck Hb, which has been renamed as RhuB (R. anatipestifer hemin uptake receptor B).

Sequence analysis of rhuB
In a previous study, RNA-seq data showed that the B739_1068 transcript is upregula ted under iron-limited conditions (23).The promoter region of B739_1068 contains a putative Fur box (7).However, sequence comparison showed that B739_1068 had low identity (<20%) with well-characterized FecA proteins, such as FecA of Escherichia coli, FecA of Pseudomonas aeruginosa, and FecA of Hafnia paralvei (data not shown).CLUSTALW analysis comparing B739_1068 with well-characterized TonB-dependent hemin receptors, including HemR of Serratia marcescens, HasR of Serratia marcescens, HpuB of Neisseria meningitidis, HmuR of Porphyromonas gingivalis, RhuR of R. anatipesti fer, HuxC of Haemophilus influenzae and ChuA of Escherichia coli, showed identity rates in the 12%-17% range.Thus, it was impossible to determine whether B739_1068 functions as an iron compound receptor or as a hemin receptor through sequence analysis (data not shown).Based on these data, as well as the data described below, B739_1068 is designated as RhuB (R. anatipestifer hemin uptake receptor B).

R. anatipestifer CH-1 gene rhuB is regulated by iron and Fur
Previous transcriptome findings have shown that B739_1068 (rhuB) in RA CH-1 is an iron-regulated gene (23).Herein, we aimed to examine whether it was also regulated by Fur.Iron-limited conditions significantly upregulated the transcription rate of rhuB compared to iron-rich conditions, and this induction was repressed by exogenous Fe(NO 3 ) 3 (Fig. 1A).The transcript level of the rhuB gene increased more than 20-fold in the fur mutant strain compared to that in the wild-type (WT) strain, and the addition of EDDHA did not affect the transcription rate of rhuB in the fur mutant strain (Fig. 1A).Moreover, the increased transcript levels were fully restored to the WT levels by complementation with fur (Fig. 1A).The negative regulatory effect of iron and Fur on rhuB was further confirmed at the protein level with the observation that RhuB production was significantly elevated in the iron-limited condition and fur mutant strain compared with that in the WT and complemented strains, respectively (Fig. 1B; Fig. S1).Sequence comparison shows that the promoter region of rhuB contains an R. anatipestifer Fur box (5′-ATTTAGAATTATCCTAAAT-3′) (7).The motif was visualized using the TBtools software (24) (Fig. 1C).To further demonstrate that the Fur protein has a direct interaction with the promoter region of rhuB (194 bp), an electrophoretic mobility shift assay (EMSA) experiment was performed as described in a previous study (7).The presence of Fe 2+ was necessary for the DNA-binding activity of Fur, but Fe 2+ is easily oxidized to Fe 3+ when exposed to air.Thus, Mn 2+ was used to substitute for Fe 2+ (7,25).As shown in Fig. 1D, the formation of DNA-Fur protein complexes was observed only when Mn 2+ was present and the binding was dependent on the concentration of Fur protein.
Using specific binding with the Hill slope equation (Graphpad prism V.9) to determine a binding affinity constant (K d ), we derived the value of 4.3 × 10 −9 M for the recombinant Fur-binding promoter region of rhuB (26).These results suggested that rhuB expression is directly regulated by Fur.

RhuB was involved in hemin utilization from duck Hb
Because RhuB, which encodes a putative TonB-dependent receptor, was regulated by iron and Fur, it was proposed that RhuB was involved in iron or hemin transportation.Thus, we identified the growth effect of RA CH-1ΔrhuB (ΔrhuB) in the iron-limited medium supplemented by Fe(NO 3 ) 3 , where inactivated duck serum or duck Hb served as the sole iron source, respectively.As shown in Fig. 2A, the mutant of rhuB did not affect the growth of RA CH-1 (WT) in a GCB medium or GCB medium containing the iron chelator EDDHA.Using 100 µM Fe(NO 3 ) 3 or 0.5% inactivated duck serum as a sole iron source, the growth of all bacteria was restored and no difference in growth rates was observed (Fig. 2A and B).Similarly, a lower concentration of Fe(NO 3 ) 3 or inactivated duck serum as the sole iron source did not lead to significant growth differences among the bacterial strains (data not shown).Meanwhile, the addition of 1 µM duck Hb restored the growth of all strains in an iron-limited medium (Fig. 2B).However, duck Hb supple mentation at 0.3 µM or hemin supplementation at 0.3 µM restored the growth of the WT and complemented strains but did not restore the growth of the rhuB mutant strain in iron-limited medium (Fig. 2C).These results suggest that rhuB is required for hemin utilization from duck Hb but is not involved in iron utilization from duck serum.

RhuB is a membranous hemin-binding protein
Because RhuB is involved in the utilization of hemin from duck Hb, it may be a hemin-binding protein.First, the subcellular distribution of this protein in RA CH-1 was determined.Membrane and cytosolic fractions of RA CH-1 were prepared, and RhuB was detected by western blotting using a specific antibody against RhuB.RhuB was detected exclusively in membrane fractions (Fig. 3A; Fig. S2).To identify the potential physical interactions between recombinant RhuB and hemin, the recombinant proteins were separated by SDS-PAGE and transferred to a nitrocellulose filter for hemin blotting.This method has been used to detect the binding of hemin to cytochrome C ( 27) and (Lane 4) and RA CH-1Δfur pLMF03::fur grown in the GCB medium (Lane 5).All samples were subjected to SDS-PAGE and RhuB was detected using a specific anti-RhuB antibody.RecA was used as the internal (Continued on next page) the ISD system of Staphylococcus aureus (28).Similar to recombinant HasA of Serratia marcescens (positive control), recombinant RhuB was able to bind to hemin (Fig. 3B).In contrast, the recombinant TonB1 protein from RA CH-1 (negative control) was unable to bind to hemin under these conditions (Fig. 3B).When the protein binds to hemin, it produces a specific Soret band (29).Therefore, hemin binding to rRhuB was assessed spectrophotometrically.As shown in Fig. 3C, recombinant RhuB (20 µM) was incubated with different concentrations of hemin (6,10,20,30, and 50 µM), resulting in the appearance of a Soret peak at 415 nm, indicating that RhuB-hemin complex formation.The inset in Fig. 3C shows the absorbance values of hemin-rRhuB minus those of hemin alone at 415 nm with increasing hemin concentrations.Plotting the spectral changes observed at 415 nm versus hemin concentration yielded a saturation curve (Fig. 3C).When the hemin concentration is less than 20 µM, the absorbance at 415 nm increases with the increase of the hemin concentration, while when the hemin concentration is greater than 20 µM, the absorbance at 415 nm does not increase (Fig. 3C), indicating that the binding of hemin to the recombinant RhuB depends on the concentration and may occur at a 1:1 molar ratio.Collectively, these findings suggested that rhuB of RA CH-1 encodes a membranous hemin-binding protein.
Furthermore, we deleted rhuB in strains RA CH-1ΔtonB1ΔrhuR pLMF03 (ΔtonB1ΔrhuR) and RA CH-1ΔtonB2ΔrhuR pLMF03 (ΔtonB2ΔrhuR), and evaluated their hemin utilization capacity.As shown in Fig. 4A, compared to ΔtonB1ΔrhuR, RA CH-1ΔtonB1ΔrhuRΔrhuB pLMF03 (ΔtonB1ΔrhuRΔrhuB) exhibited a significant growth delay in GCB supplemented with 120 µM EDDHA and 0.3 µM duck Hb.However, the deletion of rhuB did not affect hemin utilization of ΔtonB2ΔrhuR (Fig. 4B).This result suggests that RhuB functions as a TonB2-dependent hemin receptor.It further indicates that RhuB is the second TonB2-dependent hemin receptor.
In our previous study, we showed that the function of the outer membrane heminbinding protein, RhuA, is dependent on RhuR (22).We further investigated whether RhuA is dependent on RhuB.Therefore, we constructed the strain RA CH-1ΔrhuBΔrhuA (ΔrhuBΔrhuA) and evaluated its hemin utilization capacity.As shown in Fig. 4C, when duck Hb or hemin was the sole iron source, the ΔrhuBΔrhuA exhibited a signifi cant growth deficiency compared to the ΔrhuA.Conversely, there was no noticeable difference in growth rates between the ΔrhuBΔrhuA and ΔrhuB under the same condition (Fig. 4C).These results suggested that RhuA transfers hemin to RhuB.

RhuB mutant has no effect on R. anatipestifer virulence
Hemin uptake systems are required for the virulence of many bacterial pathogens (31).To investigate the role of the RhuB-dependent hemin transport system in the pathogenesis, RA CH-1 pLMF03 (WT), RA CH-1ΔrhuB pLMF03 (ΔrhuB), and RA CH-1ΔrhuB pLMF03::rhuB (ΔrhuB C ) were prepared to infect 3-day-old ducklings.As shown in Fig. 5, the survival rate of ΔrhuB was not significantly different from that of the parental strain.Moreover, at 24 h postinfection, compared with the WT, ΔrhuB had colonized the ducklings at similar levels (Fig. 5).Furthermore, we compared the pathogenicity of the WT and ΔrhuBΔrhuR double mutants in the ducklings.The results showed no significant differences in mortality rates or colonization levels between the two strains (data not shown).These data suggested that the rhuB mutant had no effect on R. anatipestifer virulence.

Conclusion
In this study, we identified a novel hemin TonB-dependent receptor RhuB, whose expression is regulated by iron and Fur protein levels.Moreover, this study showed that RhuB function is contingent on the presence of TonB2.Despite its significant role in hemin utilization, rhuB mutation did not notably affect the virulence of R. anatipes tifer, indicating the presence of other compensatory hemin acquisition mechanisms that sustain its pathogenicity.These insights into the role and regulation of RhuB in R. anatipestifer enhance our understanding of bacterial hemin utilization.

Bacterial strains, plasmids, and primers
The bacterial strains and plasmids used in this study are listed in Table S1.Primers used are listed in Table S2.

FIG 6
Systematic evolutionary tree of RhuB and its homologous proteins.The systematic evolution analysis of RhuB is highlighted in red.A systematic evolutionary tree was constructed using the neighbor-joining method in the MEGA7 software.The scale bar represents the percentage divergence (distance).
The numbers represent bootstrap values.

In vitro growth rate determination
The in vitro growth rates of the tested strains were determined by measuring the OD 600 (optical density at 600 nm) using a spectrophotometer (Eppendorf Biophotometer, Germany), as described previously (30).Briefly, cultures in the early exponential phase were inoculated in 20 mL GCB liquid medium at an OD 600 of 0.1 and incubated at 37°C with shaking (180 rpm).OD 600 was determined every 2 h for 14 h.

Preparation of cytoplasmic proteins and membrane proteins
Membrane fractions of RA CH-1 were prepared as previously described (22).Briefly, strain RA CH-1 was grown in 200 mL GCB liquid medium to an OD 600 of 1.The cells were harvested by centrifugation at 8,000×g for 10 min and washed twice with PBS.The pellet was resuspended in 30 mL 20 mM Tris-HCl (pH 7.4)-10 mM EDTA-1 mM Na-p-tosyl-L-lysine chloromethyl ketone (TLCK) and lysed using a French press (Thermo).Cell debris was removed from the lysate by centrifugation at 8,000×g for 10 min at 4°C, and the supernatant was centrifuged at 100,000 g for 2 h at 4°C; 30 mL of the supernatant (cytosol) was transferred to a new tube.The pellet was resuspended in an equivalent volume of 1% N-lauryl sarcosine Na (Lot# SLBK2574 V; Sigma-Aldrich) and incubated for 1 h at room temperature as the membrane protein.

Construction of the markerless mutant in R. anatipestifer CH-1
A series of genes (rhuB and rhuA) were deleted from RA CH-1 or R. anatipestifer derivative strains using a markerless deletion method, as described in a previous study (33).Primers used are listed in Table S2.Briefly, the upstream and downstream regions of the deleted region were amplified from the genome of RA CH-1 using the primers RhuB upP1 and RhuB upP2, RhuB downP1 and RhuB downP2, RhuA upP1 and RhuA upP2, RhuA downP1, and RhuA downP2 (Table S2).The two PCR fragments were ligated by overlapping PCR.The fused PCR fragment was cloned into the suicide vector, pOES (33).The recombinant plasmids were introduced into the CaCl 2 -competent strain E. coli S17-1 (34) and then transferred into strain RA CH-1 by conjugation, according to a previously described method (30).The transconjugants were selected from the GCB agar plates supplemented with Cfx (1 µg/mL) and Kan (50 µg/mL).The second homologous recombination was screened using 13 mM p-Cl-Phe counter selection, and the correct clone was identified as previously described (33).

Construction of complementation plasmids and strains
Complementation plasmids were constructed based on the shuttle vector pLMF03 as described previously (30).Primers used are listed in Table S2.The plasmid pLMF03 derivatives were introduced into CaCl 2 -competent E. coli strain S17-1, and then transferred to R. anatipestifer strains by conjugation and selected on the blood plate containing 1 µg/mL Cfx, as described previously for complementation studies (30).

Construction of the plasmids for protein expression
To express His-tagged recombinant RhuB, the complete RA CH-1 rhuB gene was amplified by PCR from RA CH-1 chromosomal DNA using the primers RhuB ExpP1 (introducing an NdeI site) and RhuB ExpP2 (introducing a KpnI site and His tag) (Table S2).PCR fragments were purified, digested with the corresponding restriction endonu cleases, and ligated into the plasmid pET32a, which had been digested with the same restriction endonucleases.Ligation mixtures were introduced into CaCl 2 -competent E. coli DH5α, and transformants were selected on LB plates containing Amp at 100 µg/mL.The colonies were screened by PCR using the corresponding primers.The validity of the sequence was determined using sequencing.The recombinant plasmids were transformed into E. coli BL21(DE3).

Protein expression, purification, and antibody production
Protein expression strains JP313 pBAD24::tonB1 (1), BL21(DE3) pET32a::rhuB, and BL21(DE3) pET32a::fur (7) were grown in 500 mL LB medium at 37°C to mid-log phase.Then, 0.02% arabinose (for JP313 pBAD24::tonB1) or 0.5 mM IPTG (for BL21 pET32a::rhuB and BL21 pET32a::fur) was added, and the cultures were further incubated for 3 h at 37°C before being harvested.The cultures were washed in 20 mL of binding buffer (50 mM Tris-HCl, 0.05% Triton, 250 mM NaCl, pH8.0) by centrifugation at 8,000×g for 10 min.The pellet was either stored at −20°C until use or resuspended in lysis buffer (binding buffer supplemented with 1 mg/mL lysozyme and 1 U/mL DNase I) for lysis.Cell debris was removed by centrifugation at 12,000×g at 4°C, and recombinant proteins were isolated as described in a previous study (1).Protein concentrations were determined using the Bradford assay according to the manufacturer's instructions (Bio-Rad), with Bovine Serum Albumin as the standard.The purified His-tagged recombinant protein was used to produce polyclonal antibodies in mice using standard methods described in a previous study (1).

Real-time RT-PCR
Real-time PCR was performed to evaluate the expression of rhuB in RA CH-1 cells.RNA was isolated using the RNeasy Mini Kit (Qiagen), and the isolated RNA was treated with DNase I (Qiagen) according to the manufacturer's instructions.Real-time qPCR was performed as previously described (30).Fold change was calculated as described previously (35) using the delta-delta Ct method to consider the efficiency of the PCR reaction for each target.Quantitative measurements were performed on biological samples in triplicate, and the results were normalized to the RA CH-1 housekeeping gene 16S rDNA.

Western blot analysis
Western blotting was performed as previously described (36) with slight modifications.Briefly, proteins were resolved by SDS-PAGE and transferred onto Polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA).The membrane was blocked in 5% (w/v) nonfat milk powder overnight at 4°C and incubated with primary antibodies for 2 h at 37°C.The membrane was washed three times in TBST buffer (50 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.4) and incubated with a 1:2,000 dilution of a goat anti-mouse IgG-coupled alkaline phosphatase-conjugated secondary antibody for 1 h at 37°C.Signals were detected using a BCIP/NBT solution, following the manufacturer's instructions (Sigma), or enhanced chemiluminescence reagents (GE Healthcare ECL Plus) in the ChemiDoc MP Imaging System (Bio-Rad).The RecA antibody was prepared as previously described (30).

Hemin-binding assay
The hemin-binding ability of RhuB was investigated using a previously described protocol with slight modifications (28).Briefly, proteins (1 µg) were mixed with 1 × loading buffer (200 mM Tris-HCl, 25% glycerin, 5% SDS, and 0.1% bromophenol blue, pH6.8) and separated using 12% SDS-PAGE.One gel was stained with Coomassie brilliant blue R250.Another gel was transferred to nitrocellulose, and the nitrocellulose membrane was washed for 10 min with TBST (10 mM Tris-HCl pH = 8.0, 150 mM NaCl, and 0.1% Tween 20) and subsequently probed for 1.5 h with TBS containing hemin (10 −6 M) at room temperature.The nitrocellulose membrane was washed thrice for 10 min with TBST at room temperature.Hemin was visualized for its intrinsic peroxidase activity using enhanced chemiluminescence reagents (GE Healthcare ECL Plus) on a ChemiDoc MP Imaging System (Bio-Rad).

Virulence and colonization assay
Virulence and colonization assays were performed as described previously (22).In brief, bacteria grown to an OD 600 of 1-1.5 in GCB medium were harvested, washed, and resuspended in PBS to a final concentration of 10 9 CFU/mL.Ten 3-day-old ducklings per group were injected with a 200-µL dose of 10 9 CFU bacteria in the leg using a syringe.Survival was recorded for all strains for 7 days.For the colonization assay, 3-day-old ducklings (10/group) were injected with 5 × 10 8 CFU of WT, ∆ruhB and ∆ruhB C in the leg.At 24 h post-infection, six selected ducklings were euthanized, and tissues were collected, weighed, ground, diluted, and plated on blood agar plates to determine the number of bacteria per milliliter or gram of tissue.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 9 software for Windows.Statistical significance was ascertained using Student's t-test.P-values of <0.05 were considered significant.

Bioinformatic analysis
RhuB and its homologous sequences were obtained from the NCBI database.Homolo gous proteins were selected based on an identity greater than 40% and coverage greater than 90%.Multiple sequence alignments were performed using the Cluster Omega tool (39).A phylogenetic tree was constructed by the neighbor-joining method (bootstrap replicates ×1,000) using the MEGA7 software (40), followed by subsequent refinement using iTOL v6 (https://itol.embl.de).

FIG 1
FIG 1 Expression rate of rhuB is regulated by iron and Fur levels.(A) Left, qRT-PCR analysis of the fold change in the iron-responsive transcript levels of the rhuB gene in RA CH-1 grown in the GCB medium (column 1), GCB medium containing 120 µM EDDHA (column 2) or 120 µM EDDHA together with 100 µM Fe(NO 3 ) 3 (column 3).Right, The transcript levels of rhuB in RA CH-1 pLMF03 grown in GCB medium (column 1) and in RA CH-1Δfur pLMF03 grown in the GCB (column 2) and GCB medium supplemented with 120 µM EDDHA (column 3), and the transcript levels of rhuB in RA CH-1Δfur pLMF03::fur in the GCB medium (column 4).The values shown are the averages and standard deviations derived from three experiments.Asterisks denote significant differences (*P < 0.05, **P < 0.01) between two groups; n.s, not significant.(B) Left, western blot detection of RhuB in lysates from RA CH-1 pLMF03 grown in the GCB medium (Lane 1), in GCB medium combined with 120 µM EDDHA (Lane 2), in GCB medium together with 120 µM EDDHA supplemented with 100 µM Fe(NO 3 ) 3 (Lane 3); lysates from RA CH-1Δfur pLMF03

FIG 1 (FIG 2
FIG 1 (Continued) reference.Right: RhuB protein expression in RA CH-1 derivative strains grown under different conditions.The intensity of the RhuB band was measured using ImageJ software.(C) Sequence logo of a Fur box in R. anatipestifer.(D) Left, identification of a Fur box at the rhuB promoter region, indicated in red.Right, binding of Fur to the rhuB promoter, as examined by EMSA, escalating quantities of purified His 6 -Fur protein.

FIG 3
FIG 3 Subcellular location of RhuB in R. anatipestifer CH-1 and the hemin-binding assay of the recombinant RhuB.(A) RhuB was detected in the RA CH-1 lysate, cytosol, and membrane using a polyclonal antibody.The subcellular localization of the membrane protein TonB1 in RA CH-1 cells was used as a positive control.rProtein indicates the purified recombinant protein used for comparison.(B) Left, recombinant HasA of Serratia marcescens (Lane 1; positive control), recombinant RhuB (Lane 2), and recombinant TonB1 (Lane 3; negative control) were run on a polyacrylamide gel.The gels were stained with Coomassie brilliant blue.Right, the proteins were transferred to a nitrocellulose filter and subjected to hemin blotting.The experiment was repeated three times, and independent experiments were performed.(C) Increasing concentrations of hemin were added to 20 µM recombinant RhuB, and the absorption spectrum from 250 to 700 nm was measured using nanodrop 2000.The spectrum corresponding to 50 µM hemin was used as a control.The inset shows the absorbance values of hemin-rRhuB minus those of hemin alone at 415 nm with increasing hemin concentrations.