The Bacteroidetes are a phylum of Gram-negative anaerobic bacteria that often have intimate relationships with their human or animal hosts, make up a large proportion of the natural intestinal flora and are dominated by species such as Bacteroides fragilis and Bacteroides thetaiotaomicron. Others, such as Tannerella forsythia, are associated with oral infections such as periodontal disease . However, B. fragilis is also a commonly isolated nosocomial pathogen  and is also associated with bacterial vaginosis and pre-term birth . The intimate relationship of these bacteria with their hosts is characterized by several aspects of their biology that include the ability to manipulate host metabolism [4,5] and modulate the host immune response [6,7]. In addition, this group of bacteria is characterized by possessing a wide range of glycosidase activities targeted at acquiring carbohydrate moieties both from their environment, but also directly from host glycoproteins [8,9]. Once released, the oligo- or mono-saccharide sugars are often acquired by dedicated transport systems that allow them to traverse the inner and outer membranes of these Gram-negative bacteria .
One component of the host glycome targeted by these bacteria is a family of nonulosonic acids known as sialic acids. The most prominent member of this family in humans is the 5-acetylated version known as Neu5Ac (5-N-acetylneuraminic acid). This sugar is present as the terminal moiety of a range of human glycoproteins, such as TLRs (Toll-like receptors) , integrins , mucins  and the blood group antigens SLeX/SLeA (sialyl-Lewis A/sialyl-Lewis X) . Most commonly, it is linked to underlying sugars via α-2,3 or α-2,6 glycosidic linkages that are targeted by secreted or cell-associated sialidase enzymes from several members of the Bacteroidetes and other important human commensals and pathogens [8,9]. Once released, Gram-negative bacteria must transport free sialic acid across both their inner and outer membranes for use in catabolic pathways, or for reprocessing on to their cell surface . In Bacteroidetes, catabolism initiates when sialic acid is converted into ManNAc (N-acetyl mannosamine) and pyruvate, by the action of a neuraminate lyase [NanA (N-acetylneuraminate lyase)] before an epimerase [NanE (N-acetylmannosamine-6-phosphate 2-epimerase)] converts ManNAc into GlcNAc (N-acetylglucosamine), that is phosphorylated by the RokA kinase before being turned into fructose 6-phosphate and subsequently used in glycolysis or cell wall biosynthesis .
There are several well-studied bacterial sialic acid inner-membrane transport systems, such as the SiaPQR TRAP (tripartite ATP-independent periplasmic) transporter from Haemophilus influenzae , the sodium solute symporter STM1128 from Salmonella enterica serovar Typhimurium , and the NanT MFS (major facilitator superfamily) permeases that are present in Escherichia coli and several Bacteroidetes species including T. forsythia and B. fragilis . However, much less is known about how sialic acid traverses the outer membrane. This was first studied in E. coli, where the non-specific porins OmpC (outer membrane protein C) and OmpF (outer membrane protein F), but also the sialic acid-specific porin NanC (N-acetylneuraminic acid outer membrane channel protein) allow sialic acid to traverse the outer membrane . More recently, our laboratory identified a novel sialic acid utilization operon in the T. forsythia genome that contains a putative TonB-dependent sialic acid-specific outer membrane transporter which we named NanOU . It comprises a predicted β-barrel protein of the TBDR (TonB-dependent receptor) family, NanO (neuraminate outer membrane permease), adjacent to a smaller protein, NanU (extracellular neuraminate uptake protein) that has homology with the SusD family of proteins. The SusD proteins are outer membrane-associated proteins that act to bind and sequester oligosaccharide substrates that are transported by cognate SusC TBDR proteins, homologues of NanO [19,20]. Although the SusCD system from B. thetaiotaomicron is the best studied, they are part of a larger group of polysaccharide transport systems known as a PULs (polysaccharide utilization loci), which typically comprise a TBDR, a surface-associated binding protein, and a glycosidase or other sugar-processing enzyme . Owing to this homology and a conserved genetic co-localization with sialidases, sialic acid processing and catabolism genes in Bacteroidetes species, we postulated that NanOU is a novel PUL that targets the monosaccharide sialic acid after its release from sialo-glycans attached to host surface glycoproteins. In the present study, we present genetic, biochemical and structural data that elucidates the function of NanU as a high-affinity sialic acid-binding protein that works in concert with its cognate TBDR, NanO, in sialic acid transport.
MATERIALS AND MATHODS
T. forsythia (A.T.C.C. number 43037) was routinely grown anaerobically (10% CO2, 10% H2 and 80% N2) at 37°C on FA (fastidious anaerobe; Lab M) agar plates supplemented with 5% (v/v) oxalated horse blood containing 10 μg/ml NAM (N-acetylmuramic acid) and 50 μg/ml gentamycin. Liquid cultures were grown in trypticase soy broth plus 0.4% yeast extract supplemented with 10 μg/ml NAM, 50 μg/ml gentamycin, 5 μg/ml haemin and 1 mg/ml menadione. B. fragilis (NCTC 9343; a gift from Professor Sheila Patrick, Queen's University Belfast, Belfast, U.K.) was cultured identically, but in the absence of NAM and gentamycin. B. fragilis was also cultured on a defined media agar [1.5% (w/v)] adapted from the method described by Varel and Bryant  with modifications to the base medium by the addition of 1 mg/ml haemin (in 20 mM NaOH), 10 mg/ml menadione (vitamin K3), 0.74% methionine and 0.167% cobalamin (vitamin B12) with 15 mM glucose or Neu5Ac added as carbon sources. E. coli strains were routinely grown at 37°C in LB medium or M9 minimal medium as described previously  with appropriate supplements and carbon sources as indicated. Selective antibiotics were added to the appropriate concentrations as indicated. The strains used in the present study are listed in Supplementary Table S1 (at http://www.biochemj.org/bj/458/bj4580499add.htm).
Production of ΔtonB strain and complementation plasmids
To construct a MG1655 ΔnanCnanR(amber) ΔompR::Tn10(tet) ΔtonB::FRT-Km-FRT and an MG1655 ΔtonB::FRT-Km-FRT strain, we introduced a tonB-deletion mutation into our previously constructed MG1655 ΔnanCnanR(amber) ΔompR::Tn10(tet) triple mutant and wild-type MG1655 using the gene-disruption method described by Datsenko and Wanner . Briefly, tonB mutagenesis PCR products carrying a kanamycin-resistance gene were amplified from pKD13 plasmid using the primers ECtonB-FRT-F and ECtonB-FRT-R (Supplementary Table S2 at http://www.biochemj.org/bj/458/bj4580499add.htm) and used to transform the ΔnanCnanR(amber) ΔompR::Tn10(tet) or MG1655 strain carrying the pKD46 helper plasmid with selection on LB plates with 50 μg/ml kanamycin. After curing of pKD46, the correct insertion was verified by PCR and sequencing while the phenotype of the single ΔtonB::FRT-KM-FRT mutation was confirmed by its dependency on cobalamin for growth.
To perform complementation experiments, nanOU homologues from B. fragilis NCTC 9343 were inserted into the arabinose inducible pBAD18  plasmid before transformation into the strains indicated. The open reading frames of BF-NanO (B. fragilis NanO; BF1719; GenBank® accession number CR626927.1, region 2005661–2008927), BF-NanU (BF1720; region 2008979–2010526), and BF-NanOU (BF1719-BF1720; region 2005661–2010526) with stop codons at the 3′-end and an E. coli ribosome binding site (from pET15b) at the 5′-end were PCR-amplified using Phusion high-fidelity DNA polymerase (New England Biolabs) and the primer pairs BFO-XbaI-F/BFO-HindIII-R, BFU-XbaI-F/BFU-SphI-R and BFO-XbaI-F/BFU-SphI respectively (Supplementary Table S2 at http://www.biochemj.org/bj/458/bj4580499add.htm). The amplicons were inserted into pBAD18 via either XbaI-HindIII or XbaI-SphI doubly digested vector and inserted where appropriate. Along with the pBAD18 empty vector, the sequence-verified (Core Genomic Facility, University of Sheffield, Sheffield, U.K.) cloned constructs, named pCP-cBFO, pCP-cBFU, pCP-cBFOU and pCP-cTFOU, were used in the transformation of electrocompetent E. coli MG1655 ΔnanCnanR(amber) ΔompR::Tn10(tet) and ΔnanCnanR(amber) ΔompR::Tn10(tet) ΔtonB::FRT-Km-FRT strains, with selection on LB medium with 50 μg/ml ampicillin. Parental, mutant and the generated single/double-complemented strains were pre-cultured at 37°C overnight in M9 liquid medium containing 15 mM glucose as the carbon source. Strains with the ΔtonB::FRT-Km-FRT mutation were additionally supplemented with 88 μM iron (provided as FeCl3·6H2O) and 0.005 mM cobalamin. Stationary-phase cells were washed with sterile M9 medium, and used to inoculate fresh M9 medium with 0.5, 1, 3, 6 or 15 mM carbon source (glucose, Sigma–Aldrich, or Neu5Ac, Carbosynth) to a final A600 of 0.05 without induction with arabinose. Minimal medium growth experiments were carried out at 37°C, with absorbance measured (at A600) over time. Results are means of these three experiments and significant differences assessed using Student's t test.
Production of recombinant NanU
The entire ORF of BF-nanU (BF1720) was PCR-amplified using Phusion high-fidelity DNA polymerase (New England Biolabs) from the genomic DNA of B. fragilis NCTC 9343 using the primers BFnanU-NdeI-F and BFnanU-XhoI-R (Supplementary Table S2). The amplicon was then inserted into the NdeI and XhoI sites of pET21a(+) (Merck Millipore) and verified by sequencing (Core Genomic Facility, University of Sheffield). The coding region of TF-NanU (T. forsythia NanU; GenBank® accession number CP003191.1, region 2360849–2362417) was synthesized in an E. coli codon-optimized construct by Eurofins MWG such that changes in codon usage resulted in no changes in the primary amino acid sequence. The gene was subcloned from the holding plasmid pEX-A into pET21a(+) using NdeI and XhoI, and its sequence was confirmed by DNA sequencing. E. coli BL21λ(DE3) cells were transformed with plasmids and grown in either LB or M9 (glycerol) broth-cultured cells with induction (1 mM IPTG; Sigma–Aldrich) during the mid-exponential growth phase (A600=0.6) for 5 h at 37°C with agitation. Cells were harvested and resuspended in 25 mM sodium phosphate, pH 7.4, 0.5 M NaCl and 25 mM imidazole, disrupted using a French pressure cell (Thermo Scientific) and soluble fractions clarified by further centrifugation (10000 g for 30 min at 4°C). The C-terminally His6-tagged proteins were purified by Ni2+–Sepharose affinity chromatography and eluted with a 50–200 mM imidazole gradient on the ÄKTAprime plus system (GE Healthcare). The purified proteins were extensively dialysed against 25 mM sodium phosphate, pH 7.4, concentrated using a MWCO (molecular-mass cut-off) 10000 Vivaspin column (GE Healthcare) and protein concentrations were determined using the Pierce BCA protein assay kit (Thermo Scientific). The identities of recombinant BF-NanU (BF1720) and TF-NanU (TF0034) were confirmed by LC–MS/MS (ChELSI, University of Sheffield).
Sialic acid-binding studies
The binding characteristics of purified NanU with candidate ligands were investigated by steady-state tryptophan fluorescence spectroscopy [BF-NanU (BF1720) and TF-NanU (TF0034) possess seven and eight tryptophan residues respectively]. Proteins were diluted in 25 mM sodium phosphate buffer, pH 7.4, to a final concentration of 0.5 μM and incubated at 25°C for 5 min before substrate titration. The quenching of intrinsic tryptophan fluorescence was measured at 25°C with constant stirring in a 104F-QS quartz fluorescence semi-micro cell (Hellma Analytics) using a Varian Cary Eclipse spectrofluorometer. The excitation and emission wavelengths were 295 nm (5 nm slit width) and 330 nm (10 nm slit width) respectively. Corrections for background fluorescence changes due to dilution effects with the stepwise addition of ligands were made by buffer titrations. Analysis and curve fitting of titration data were performed in Prism 5 (GraphPad Software), with the equilibrium dissociation constants (Kd) for one site-specific binding calculated using eqn (1): (1) where X is the final concentration of ligand, Y is the percentage increase in protein quenching in relation to the protein-only fluorescence signal and Bmax is the extrapolated maximum specific binding.
Crystallization and data collection of BF-NanU
Purified BF1720 was concentrated to 6 mg/ml in 25 mM sodium phosphate, pH 7.4, and tested for crystallization at 3 mg/ml with a variety of commercial screens. Subsequent optimization resulted in the growth of X-ray diffracting crystals grown with 1 μl of protein and 1 μl of reservoir solution [0.2 M ammonium acetate and 20% (w/v) PEG 3350] after 14 days of incubation at 17°C. Crystals were mounted direct from the drop without the addition of cryoprotectant and flash-cooled in a nitrogen gas stream maintained at 100 K. Data were collected to 1.6 Å on station I04-1 at the Diamond Light Source (Harwell, U.K.), and processed using XDS . Molecular replacement was performed using the program PHASER on these data, employing a model obtained from the PHYRE2 server  based upon a SusD superfamily protein from Bacteroides vulgatus (PDB code 3JQ0). The initial model was improved by application of the PIRATE and BUCCANEER programs from the CCP4 suite  coupled to manual intervention in COOT  interspersed with rounds of refinement using REFMAC . A summary of the relevant data statistics is shown in Supplementary Table S3 (at http://www.biochemj.org/bj/458/bj4580499add.htm). Structural factors and co-ordinates have been deposited in the PDB under code 4L7T. All Figures were generated using PyMOL (http://www.pymol.org) and the topology diagram was prepared using CorelDraw X4 (Corel Corporation).
To ascertain whether both native and ligand-bound BF1720 were monomeric, 3 mg/ml of purified protein with and without pre-incubated Neu5Ac in 25 mM sodium phosphate, pH 7.4, at an equimolar concentration were sequentially applied to a HiLoad Superdex 200 PG gel-filtration column (GE Healthcare) at a flow rate of 1 ml/min. Apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), ovalbumin (43 kDa), trypsin inhibitor (20 kDa), cytochrome c (12.3 kDa) and aprotinin (6.5 kDa) were used as molecular mass standards.
Production of an anti-NanU antibody and the localization of NanU
To produce a polyclonal antibody specific against BF1720, purified protein was injected into rats and the animals were subsequently boosted twice with the same antigen (BioServ U.K.). The specificity of the antisera was confirmed in test blots on the lysates of B. fragilis, E. coli, T. forsythia and Porphyromonas gingivalis, with only the NanU band highlighted in its native species and no cross-reacting bands present in the other three species even at high antisera titres (results not shown).
Preparation of cellular and secreted fractions from Bacteroidetes cultures
Whole-cell samples were normalized according to the absorbance, i.e. 1 ml at A600=1.0 was resuspended in 200 μl of Laemmli sample buffer and 10 μl subjected to SDS/PAGE (10% gel). Cells were fractionated in a method adapted from previous work . B. fragilis NCTC 9343 and T. forsythia A.T.C.C. 43037, plate-grown in an anaerobic atmosphere for 1 and 5 days respectively, were resuspended in 25 mM sodium phosphate, pH 7.4, and 0.5 M NaCl, placed on ice and disrupted by sonication using a Soniprep 150 (MSE). Cell debris was removed by two rounds of centrifugation (5000 g for 30 min at 4°C), and fractions containing membrane proteins were pelleted (330000 g for 3 h at 4°C). The supernatants were carefully decanted and stored (−20°C) as cytoplasmic fractions. Membrane pellets were washed twice in 25 mM sodium phosphate, pH 7.4, and 0.5 M NaCl, resuspended in 1 ml of the same buffer and an equal volume of 2% (v/v) sodium N-lauroyl sarcosinate in phosphate buffer (pH 7.4, 0.5 M NaCl) added, followed by incubation at 37°C for 30 min. The suspensions were centrifuged (10000 g for 30 min at 4°C), and the supernatants containing the inner membrane fractions stored (−20°C). The outer membrane pellets were washed twice in the same phosphate buffer before resuspending in 500 μl of the same buffer and stored at −20°C. To obtain fractions containing secreted proteins, the same B. fragilis and T. forsythia strains were cultured in 20 ml of liquid broth for 1 and 5 days respectively. Cells were removed by centrifugation (10000 g for 30 min at 4°C) before the addition of 5 ml of TCA (trichloroacetic acid) to each clarified supernatant and incubation at 4°C for 15 min. Proteins were pelleted by centrifugation (10000 g for 30 min at 4°C) and the resulting pellets washed three times with 500 μl of ice-cold acetone. The pellets were dried on a 95°C heat block for 10 min before resuspension in 500 μl of Laemmli buffer. Cell fractions were resolved by denaturing SDS/PAGE (10% gel), transferred on to nitrocellulose membranes (Millipore), blocked overnight with TBS-T (TBS, pH 7.4, containing 0.05% Tween 20), 5% (w/v) skimmed milk and 3% (w/v) BSA. The membranes were then probed with BF1720 rat antiserum before incubation with HRP (horseradish peroxidase)-conjugated goat anti-(rat IgG) (Sigma–Aldrich). Proteins were visualized using a Pierce enhanced ECL HRP substrate (Thermo Scientific) before incubation with CL-XPosure X-ray film (Thermo Scientific) and development using the Compact X4 automatic film processor (Xograph Healthcare). To detect the presence of possible cytoplasmic protein contamination, Western blots were performed as described above, but using anti-(E. coli GroEL rabbit IgG) (Sigma) and anti-(rabbit HRP-conjugated goat IgG) (New England Biolabs) as the primary and secondary antibodies respectively.
Poly-L-lysine coverslips (BD BioCoat) were briefly immersed in a PBS suspension of B. fragilis cells at A600=0.1 plate-cultured for 16 h, following which the cells were fixed with 4% (v/v) paraformaldehyde in PBS (buffered to pH 7.0) for 30 min at 37°C, blocked with PBS containing 3% (w/v) BSA for 1 h and incubated with BF1720 rat antiserum (1:5000 dilution) for 2 h. The coverslips were incubated in the dark with PBS containing Alexa Fluor™ 488 goat anti-(rat IgG) (1:3000 dilution; Life Technologies) for 1 h, and mounted on to glass slides with ProLong Gold antifade reagent with DAPI (Life Technologies). Fluorescence was visualized under a Zeiss Axiovert 200 M microscope at ×100 magnification. Repeated washes of coverslips with PBS were carried out between steps, and sample preparations and visualization were carried out at 25°C unless stated otherwise.
The nanOU homologues from B. fragilis restored sialic acid-dependent growth to an E. coli ΔnanCRΔompR mutant
As published previously by our group , several genomes of species in the Bacteroidetes group contain putative homologues of the nanOU putative sialic acid-transport genes, including those from T. forsythia and also several other human pathogens and commensal species. Among these, the genes with highest homology with nanOU from the oral pathogen T. forsythia are those from the Gram-negative anaerobe B. fragilis NCTC 1943, namely BF1719 (BF-nanO) and BF1720 (BF-nanU), which have 69.0% and 63.1% amino acid identity respectively. To investigate whether the putative nanOU genes from B. fragilis functioned in a similar manner to those from T. forsythia in sialic acid transport and to begin to establish whether they might form part of a novel family of sialic acid transporters, we inserted BF-nanOU into the pBAD18 expression plasmid and transferred them into an E. coli ΔnanCnanR(amber) ΔompR::Tn10(tet) strain, which is devoid of all outer membrane sialic acid porins and is therefore unable to grow with sialic acid as a sole carbon and energy source . We then assessed its ability to grow in M9 minimal medium with sialic acid as a sole carbon source. In the present study, no arabinose was added, as our previous experience is that complementation is achieved from these plasmids without induction . In a similar manner to the TF-nanOU genes, the BF-nanOU genes were able to restore the sialic acid growth defect of this strain (Figure 1A). These data indicate that BF-nanOU not only encodes a functional sialic acid transport system, but also confirm that this may be a common sialic acid transport method in this group of important human-associated bacteria.
(A) Growth kinetics of E. coli MG1655 ΔnanCnanR(amber) ΔompR::Tn10(tet) (Δnan) mutant strains complemented in trans with the nanOU genes from T. forsythia (TF-nanOU, ○), B. fragilis (BF-nanOU, ∆) or the individual B. fragilis nanO (BF-nanO, ◇) or nanU (BF-nanU, ▼) expressed from pBAD18 (also included as a negative control, □) were monitored (A600) during culture at 37°C in M9 minimal media with 15 mM Neu5Ac. Results are means±S.D. from three separate biological replicates. (B) The Δnan strain complemented with either B. fragilis nanO (BF-nanO, open symbols) or nanOU (BF-nanOU, close symbols) was incubated as above but with 6 (● and ○), 3 (◆ and ◇), 1 (■ and □) or 0.5 (▲ and ∆) mM Neu5Ac. Results are means±S.D. from three separate biological replicates. (C and D) The sialic acid transport and tonB-deletion mutant strains [ΔnanCnanR(amber) ΔompR::Tn10(tet) ΔtonB::FRT-Km-FRT–ΔnanΔton] were complemented as in (A) and (B) in M9 medium with either 15 mM glucose (C) or Neu5Ac (D) as indicated and growth followed over time. Results are means±S.D.
NanU enhances NanO function
In order to determine the contribution of the individual nanO and nanU genes to sialic acid transport, we cloned the individual genes from B. fragilis NCTC 9343 into pBAD18, and tested their ability to restore the sialic acid growth defect of the ΔnanCnanR(amber) ΔompR::Tn10(tet) strain, again without the addition of arabinose. As shown in Figure 1(A), when BF-nanU was provided in trans with 15 mM sialic acid in the growth medium, no restoration of the growth defect was observed, but when BF-nanO was provided in isolation, growth was restored to approximately wild-type levels. To probe further the role of NanU, we asked the question of whether at low Neu5Ac concentrations, nanU enhances nanO function. To address this question we repeated the experiment at a range of lower concentrations (0.5–6.0 mM) and as shown in Figure 1(B). We consistently observed a slight, but significant, increase in the length of the lag phase of the growth curve that is most prominent at 3 mM (i.e. the curve shifted to the right-hand side) as evidenced by lower A600 readings for the nanO-only strains at all time points between 6 and 22 h (P<0.05; one-tailed Student's t test), but also at 6 mM sialic acid. In addition, we observed a lower final growth yield at lower concentrations of sialic acid, i.e. when only 1 mM sialic acid was provided (A600=0.2 compared with 0.1) in comparison with the provision of BF-nanOU in concert. We consider it unlikely that these differences are due to differences in expression levels from these plasmids, since the promoter sequences are identical for the BF-nanO and BF-nanOU constructs and there was no difference in levels of inducer added (i.e. none). Thus the data indicate a dependence on both NanO and NanU for maximal activity in supplying sialic acid to internal catabolic pathways.
NanOU activity requires a functional TonB–ExbB–ExbD system
As mentioned above, the NanOU system appears to be the first TonB-dependent system adapted for sialic acid transport. As a result, we wished to confirm the dependence of the observed sialic acid outer membrane transport by the NanOU system on the presence of a functional TonB–ExbB–ExbD complex. To achieve this, we first constructed an E. coli ΔnanCnanR(amber) ΔompR::Tn10(tet) ΔtonB::FRT-Km-FRT (referred to as ΔnanΔtonB in Figures 1C and 1D) strain by inserting a kanamycin-resistance cassette in place of tonB in the existing ΔnanCnanR(amber) ΔompR::Tn10(tet) background . When the growth of this strain on glucose was tested, it was unaffected by this mutation or the presence of the nanOU genes from T. forsythia or B. fragilis (Figure 1C). In contrast, there was no growth of this strain when sialic acid was provided as the sole carbon source, even in the presence of the B. fragilis or T. forsythia nanOU genes in trans (Figure 1D), indicating that the function of nanOU is TonB-dependent.
To exclude the possibility that TonB may have a role in sialic acid uptake itself in E. coli, we constructed a ΔtonB::FRT-Km-FRT strain (using the same primer set) in the isogenic wild-type E. coli MG1655 background. As shown in Figure 2, both the wild-type and ΔtonB strains were able to grow well on both glucose and sialic acid as a carbon source, indicating that the function of nanOU is dependent on the presence of a functional TonB–ExbB–ExbD complex.
The ΔtonB strain was grown in M9 medium containing 15 mM Neu5Ac and growth followed over time. Results are means±S.D. from three separate biological replicates.
NanU expression is altered by the presence of sialic acid
To investigate the conditions under which NanU might be expressed, we examined its expression profile in whole cells grown in defined medium. We used our anti-NanU antibody as a probe in Western blot analysis (Figure 3) with cell mass normalized by cell number and blots probed with an anti-GroEL antibody as a loading control. When B. fragilis was grown on defined medium agar plates with glucose as the carbon source, we were unable to detect NanU in the whole-cell fractions. In contrast, when grown on the same defined medium containing sialic acid in place of glucose, expression was detected strongly. Similarly, when cells grown on glucose were transferred on to sialic acid-containing plates, we again observed an increase in NanU expression by sialic acid. In addition, we observe strong expression of NanU on rich FA blood agar plates, where the blood cells probably act as a source of sialic acid. Taken together, these data indicate strong sialic-dependent NanU expression in B. fragilis.
B. fragilis NCTC 9343 cells cultured overnight on FA agar were subcultured on FA agar (16 h) (lane 4) or minimal medium agar supplemented with 15 mM glucose (lane 1), Neu5Ac (lane 2) or glucose (16 h) then Neu5Ac (16 h) (lane 3). Normalized amounts of proteins were stained with Coomassie Blue or probed with rat BF-NanU antiserum or rabbit anti-(E. coli GroEL) as described above. Molecular mass is given on the left-hand side in kDa.
NanU is an outer membrane-associated protein
Both bioinformatic predictions (presence of Sec-dependent signal sequence and similarity to SusCD protein systems) and functional studies, i.e. dependency on TonB, indicated that the NanOU proteins might be associated with the outer membrane in both T. forsythia and B. fragilis [18,30]. To test the localization of NanU, we performed a differential detergent-based cell fractionation and examined the fractions by SDS/PAGE and Western blotting using a BF-NanU antiserum, which we generated from purified His6–NanU from B. fragilis. These data clearly show that NanU is associated with the outer membrane of B. fragilis, but is not found in the cytoplasmic or inner membrane fractions (Figure 4A). In addition, we performed a control using a commercially available anti-GroEL antibody, which was raised against the E. coli GroEL protein, but which has 76% primary amino acid sequence similarity with GroEL from B. fragilis and reacts with a 60 kDa protein found only in the cytoplasm of the intracellular fractions (Figure 4A).
Henry David Thoreau once said, “If you would convince a man that he does wrong, do right. However, do not care to convince him. Men will believe what they see. Let them see.” In terms of academic writing, this phrase means being able to draw visual images with the help of words. What is an illustration essay? An illustration essay is what best describes a paper written to create a picture in the reader’s mind and deliver the target message more effectively. In this article, we will discuss the meaning, topic, and several examples of the illustration essay.
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Illustration Essay Definition & Usage
A student may ask, “What is an illustration essay”? It is a logical question. This genre of writing is rare compared to argumentative, persuasive, compare & contrast, or narrative papers. Illustration essay interprets specific situation/person/object by providing certain examples and different details to let the reader understand the selected topic broader. To understand different types of academic essays better, you may like the website full of free essay examples.
Here is the list of illustrative essay examples. Choose the topic without conducting research!
- Use specific sports terms to illustrate how to swim a stroke in Olympic swimming, dive, or demonstrate other abilities in the water.
- Explain how dancing/acting on the stage is different from the high school cheerleading; what is a higher art?
- Discuss why a sports team of your preference (basketball, football) is underrated; why the soccer team you dislike is overrated.
- Illustrate the stages a college applicant should take in writing a winning college entrance essay to join the target educational institution.
- Share how you managed to survive your first year in college with your readers by writing several effective tips from your experience.
- Explain how you used to flirt with the opposite gender correctly to avoid being a part of the “friendship zone.”
Work & Career
- Show the way professional scientists conduct research by describing every required step in details.
- Explain what an HR manager does; write down several examples from your personal interaction with the representatives of this profession.
- Illustrate what a chief from the prestigious restaurant downtown does to cook the dish of the day (e.g., a deer).
- List & explain the features of a good business writing (e.g., make a list of the winning professional terms/keywords, which helped you to pass a job interview).
- Write about the city, which used to survive some natural disaster (an earthquake, tsunami, tornado, hurricane, etc).
- The topic on how society can support children who became the victims of school bullying/hate crimes/home violence.
How to Write an Illustration Essay
Writing an essay is made of five basic steps. Before writing your paper, decide on the most effective title.
Step 1: Identify the object of your writing (a.k.a. the main illustrative essay topic) and write a powerful thesis statement, which will impress both the teacher and entire reading audience. Help your reader to understand your topic ahead. Pick minimum three keywords/points to explain why you believe/deny the specific idea - this sentence is your thesis statement.
Example: “Gender stereotypes exist in the professional world. Many business companies prefer having men as their CEOs.”
Step 2: You are almost done with your introduction paragraph. Keep on writing what you are going to share with the reader, and provide reasons for choosing a particular topic. Start the first paragraph with the hooking sentence. Several types of the hooks exist to consider: direct quote, poetry line, metaphor, simile, joke, fact, etc. This technique will grab the reader’s attention from the first line of the essay. [Learn here how to write an analytical essay]
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Step 3: Your illustration essay should be supported by the good outline (an essay outline serves as the action plan for your writing from cover to cover). Keep on writing a paragraph supporting each reason why you chose a specific essay topic until you get three good reasons.
Example: “Last time I attended a job interview, I have lost my place to Mr. Green, and the only ‘good’ reason the local HR told me is he believes women do not possess powerful marketing ideas to help their company”.
Step 4: It is the easiest step in the essay writing. The writer must list three points explaining why he chose the specific illustration topic/example at the beginning of each sentence, and then support it with the meaningful evidence retrieved from the research.
Step 5: After writing a conclusion, a professional writer would like to double-check the entire essay for the following mistakes:
- Grammar & spelling
- Plagiarism & other small issues
Even if you know how to write an illustration essay perfectly, do not ignore the stage of proofreading & editing, or hire professional online editors to check your final paper.
Types of Examples You May Use to Support an Illustration Essay Thesis
To answer, “what is an illustration essay,” the student must realize the importance of examples taken from personal experience. You should support an illustration essay with the vivid examples from your personal experience. Use several good methods to get inspired: personal observation, interviews, experience, & media.
A personal observation requires observing different locations related to your chosen topic. Do not forget to take notes explaining your impressions through five human senses.
An interview means having a face-to-face conversation with people who are experts in the fields connected with your topic. These people can share exciting examples so that your writing will stand out from the rest of the papers. Conduct a research to prepare a list of related questions before contacting the people of your interest.
Recall your personal experience to include in your writing. Personal memories are a good source of ideas you can share with the readers to support the main argument. Research & look at some images to jog your memory. Write every topic detail you remember from your personal life experience; do not forget to include sensory expressions & comments from other people. Let the adjectives and adverbs help you with your writing.
Media is one of the most useful sources of ideas & examples in the modern world. Spend some time on social networks (Facebook, YouTube, Instagram) where people of all types share their experience by writing meaningful posts or publishing interesting videos. Pick the best topic examples for your illustration essay from the following sources:
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