APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Flagellin gene (fliC )of Thermus thermophilus HB8:
characterization of its product and involvement to flagella assembly and microbial motility
Christos P.Papaneophytou &Rigini M.Papi &
Anastasia A.Pantazaki &Dimitrios A.Kyriakidis
Received:22September 2011/Revised:16January 2012/Accepted:19January 2012/Published online:23February 2012#Springer-Verlag 2012
Abstract Thermus thermophilus HB8flagellin protein (FliC)is encoded by the TTHC004(fliC )gene,which is located in the pTT8plasmid of the bacterium.Flagellin monomer and flagella fibres were isolated from a culture of T.thermophilus grown in rich medium,or in mineral salt medium with sodium gluconate as the carbon source.Western blot immunodetection with anti-FliC revealed a stable complex (FliC)1(FliS)2of flagellin (FliC,27.7kDa)with a homodimer of FliS (FliS,18.2kDa)that are encoded by TTHC004and TTHC003genes,respectively.The complex is dissociable at low pHs and/or by heat treatment.Glycan staining of purified flagella and treatment with N -glycosidase F suggested that flagellin of T.thermophilus is a glycosylated protein.Size exclusion chromatography revealed that flagellar filaments (FliC)have a molecular mass higher than 200kDa.The formation of flagella is enhanced after prolonged cultivation time where phosphate and other nutrient were depleted,giving in the bacterium considerable swimming motility in low viscosity media.
Introduction
Bacteria are capable of sensing environmental changes through highly adaptive signalling machinery.They are able to direct their chemotactic movement to more favourable environments by changing their motility or altering gene expression and other cellular activities (Gluch et al.1995).Flagella are one
of the most complex and effective structures,responsible for the motility of numerous bacteria species (Nishihara and Freese 1975;Macnab 1999).They are able of propelling bacteria through liquids (swimming)and through viscous envi-ronments,or over surfaces (swarming)(Manson et al.1998).The number and arrangement of flagella vary among species.Many bacteria express a polar flagellum for swimming,while other express lateral flagella for swarming.A limited number of bacteria possess dual flagella systems and are able to express two distinct flagella systems depending on the environmental conditions (Merino et al.2006).
In contrast to unicellular swimming in liquid cultures,swarming is characterized by a multicellular movement of bacteria that migrate on solid surfaces in groups of strongly bound cells (Henrichsen 1972;Harshey 1994).Moreover,swarming is not a starvation response,like other processes of differentiation in bacteria,and therefore,it varies depending on the microorganism and the environmental growth conditions (Harshey 1994).
The flagellum is a complex structure consisting of a basal body,a trans-membrane rotary motor,a universal joint-hook structure and a helical filament.This filament has an outer diameter of 12–25nm,a 2–3-nm-diameter inner channel and can be 1–15nm or more in length (Malapaka and Tripp 2006).The flagellar basal structure is engaged in protein secretion,before serving as the flagellar motor (Komoriya et al.1999).The flagellar export system possesses similarities to the type III family (Macnab 2004).The export substrates of type III secre-tion systems do not contain cleavable signal sequences,and they do not share common sequence motifs responsible for export (V egh et al.2006).
The filament,accounting for 98%of the organelle mass,is composed exclusively of flagellin,which may comprise as many as approximately 20,000flagellin subunits (V egh et al.2006;LaV allie and Stahl 1989).Flagellin proteins are synthe-sized in the cytoplasm as soluble monomers and are prevented
C.P.Papaneophytou :R.M.Papi :A.A.Pantazaki :
D.A.Kyriakidis (*)
Laboratory of Biochemistry,Department of Chemistry,Aristotle University of Thessaloniki,54124Thessaloniki,Greece e-mail:kyr@chem.auth.gr
Appl Microbiol Biotechnol (2012)94:1265–1277DOI 10.1007/s00253-012-3913-7
Keywords T.thermophilus HB8.fliC gene .Flagellin .Flagella assembly .Motility
from self-assembly and protected from proteolytic degradation by binding of the FliS chaperon protein(Ozin et al.2003; Auvray et al.2001).The primary(phase1)flagellin gene in Salmonella typhimurium is referred to as fliC,a secondary (phase2)flagellin gene is named fljB,while other bacterial flagellin genes are flaA,hag and flaF(Malapaka and Tripp 2006).Depending on the bacterial species,flagellins have molecular masses ranging from28to80kDa(Winstanley and Morgan1997).Flagellin is polymerized to form the fila-ments of bacterial flagella and may have a role in translocation, secretion or assembly of the flagellum.
Thermus thermophilus HB8belongs to the Deinococcus–Thermus phylum and is a thermophilic bacterium with an optimum temperature range of65°C to72°C(Brock1994). The complete genomic sequence of T.thermophilus HB8has been recently determined(Henne et al.2004)(NCBI accession numbers NC_006461,NC_006462and NC_006463)and showed to be composed of1.85Mbp chromosomal DNA, 0.26Mbp plasmid pTT27and9.32kbp plasmid pTT8,encod-ing1,973,251and14open reading frames(ORFs),respec-tively.However,most ORFs of the genomic sequence of T. thermophilus HB8encode hypothetical proteins(Lioliou et al. 2004).Although Thermus bacterial cells are characterized as either immotile or possess flagella and are motile(Huber and Stetter2004),there is no reference yet for the existence of flagella in T.thermophilus,or for their motility.
The aim of this work was to investigate whether T.thermo-philus HB8is able to form flagella dependent of nutrients in the medium and thereby its potential effects on the motility of this microorganism.The gene TTHC004located in plasmid pTT8, encoding FliC,was identified.The biochemical properties of flagellin monomer(FliC)and flagella fibres isolated from a culture of T.thermophilus containing mineral salt and sodium gluconate were studied.It was revealed that flagellin formed a stable complex with FliS which is encoded by the TTHC003 gene in a ratio of(FliC)/(FliS)2that was dissociated under acidic conditions and/or thermal treatment.Size exclusion chro-matography revealed that the isolated flagella have a molecular mass of higher than200kDa.Our results indicated that T. thermophilus cells are able to swim in liquid or semisolid media after24h of cultivation where phosphate concentration was depleted.The swimming ability of T.thermophilus was attrib-uted to the formation of flagella filaments which is enhanced under these conditions.
Materials and methods
Reagents
Agar(gellam gum)was obtained from Roeper GmbH (Hamburg,Germany).Anti-flagellin antibody(anti-FliC; Salmonella H antiserum)was obtained from Difco (Franklin Lakes,NJ,USA).All secondary antibodies were purchased from Molecular Probes.The fluorescent glyco-protein detection kit(Glycoprofile III),the N-glycosidase F (Glycoprofile II)and all other reagents used were purchased from Sigma(St.Luis,MO,USA).
Bacterial strain and cultivation conditions
T.thermophilus HB8DSM579(from Deutsche Sammlung Mikroogranismen)was used in this study.To determine the effect of culture medium composition on flagella formation in T.thermophilus,two different media were tested.T. thermophilus HB8was grown in a rich medium(DSMZ-74)containing per litre:8g tryptone,4g yeast extract and 2gNaCl.The strain was also cultivated in mineral salt medium(MSM)supplemented with1.5%(w/v)sodium gluconate as sole carbon source(Pantazaki et al.2003).A 5%(v/v)inoculum of a fully grown culture of T.thermophi-lus in DSMZ-74was performed in2-l Erlenmeyer flasks containing600ml medium and subsequently incubated on a rotary shaker at70°C for72h.Aliquots of10ml were obtained at different time intervals and centrifuged at 6,000×g for15min.Cell pellets were lysed with sonication (20kHz)for30repeating cycles of20s with a20-s cooling intervals between cycles.
Motility assays
Swimming motility was assessed in cells grown in DSMZ-74or MSM agar plates containing0.3%(w/v)agar.Agar plates were inoculated with2μl from an overnight liquid culture of T.thermophilus in DSMZ-74(OD600~1)and incubated at70°C.The diameters of halos due to bacteria migration were recorded after36h of cultivation.For the swarming assay,DSMZ-74or MSM agar plates containing 1.1%(w/v)agar were inoculated with2μl from an overnight culture of T.thermophilus in DSMZ-74(OD600~1),and the plates were incubated at70°C for72h.
Isolation of flagella from semisolid agar media was per-formed by harvesting cells with cold water(Calvio et al. 2005).Cell suspensions were vortexed and centrifuged at 5,000×g for15min at4°C.Flagellar filaments were collected from supernatants by ultra-centrifugation(DuPont-SORV AL ULTRA80)at100,000×g for1h and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS–PAGE)and Western blot analysis using anti-FliC antibody. Motility assays were repeated three times on separate days.
Concentration of flagellin from the extracellular medium by TCA
The culture supernatant fraction containing total extracellular flagellin was precipitated according to LaVallie and Stahl
(1989)with some modifications.The cell-free supernatant was mixed with pre-chilled50%(v/v)trichloroacetic acid (TCA)to a final concentration of6%(v/v),incubated on ice for30min and centrifuged at12,000×g for10min.The pellet was washed twice with cold acetone and lyophilized to dryness.
Isolation of flagella fibres and other filamentous structures For the isolation of flagella fibres and other filamentous structures,T.thermophilus was grown in500ml MSM, containing sodium gluconate as sole carbon source,at70°C for72h.Cells were collected by centrifugation at6,000×g for 20min at4°C,suspended in100ml of50mM phosphate buffer,pH7.0,containing10mM MgCl2(Totten and Lory 1990)and blended in a Waring Blendor for30s.Cells were separated from flagella by centrifugation at12,000×g for 40min.Flagella were pelleted from the supernatant by ultra-centrifugation at100,000×g for1h and re-suspended in1ml of50mM phosphate buffer,pH7.0.
Flagellum staining
T.thermophilus cells were grown in DSMZ-74and in MSM supplemented with1.5%(w/v)sodium gluconate to an op-tical density at600nm of ca.0.6to0.8.A0.5-ml volume of bacterial culture was gently mixed with0.1ml of fresh fixative solution(1ml of16%paraformaldehyde,1.5μl of glutaraldehyde)and20μl of1M sodium phosphate buffer (pH7.4),as described previously(Rosu et al.2006).A20-μl volume of each sample was added into each well of a poly-l-lysine(Sigma)multitest slide and was further pro-cessed as described(Rosu et al.2006).The anti-FliC anti-body(Difco Salmonella H antiserum)and the fluorescein isothiocyanate(FITC)-conjugated goat anti-rabbit second-ary antibody were used at a1:200dilution in bovine serum albumin–phosphate-buffered saline.Samples were visualized in a Leica SP confocal microscope equipped with a×100 objective lens.
Size exclusion chromatography
Isolated flagella were loaded onto a pre-equilibrated Sephadex G200column(150×1cm,Pharmacia)in 50mM phosphate buffer,pH7.0,with a flow rate of 0.1ml/min.Eluted proteins were precipitated by adding pre-chilled50%(v/v)TCA to a final concentration of6% (v/v),incubated on ice for30min and centrifuged at 12,000×g for10min.The pellet was washed twice with cold acetone and resuspended in SDS-loading buffer.The column was size-calibrated using blue dextran(2,000kDa), alcohol dehydrogenase(150kDa),bovine serum albumin (67kDa),ovalbumin(43kDa),carbonic anhydrase (29kDa),cytochrome c(12kDa)and CrO3-(300Da). Protein determination and SDS–PAGE
The concentration of protein was determined by the Bradford (1976)method.Proteins were separated by electrophoresis in 12%(w/v)SDS–PAGE as described previously(Laemmli 1970).When indicated,proteins were subjected to electropho-resis under native conditions.
Western blot analysis
Immunoblots were performed as described previously (Burnette1981).Samples were run on a12%(w/v)SDS–PAGE and electro-transferred in transfer buffer onto nitro-cellulose or polyvinylidene fluoride membranes probed with anti-FliC antibody.
Cloning of the flagellin encoding gene
PCR amplification of flagellin encoding gene was per-formed in a50-μl reaction that contained30pmol of each primer,400μM of dNTPs,50ng of T.thermophilus HB8 genomic DNA(TAKARA BIO INC.),5%(v/v)DMSO and 3.0U of Pfu polymerase(PROMEGA).According to the supplier(TAKARA),T.thermophilus HB8genomic DNA preparation can contain the chromosomal DNA(1.85Mbp), a0.26-Mbp plasmid pTT27and finally a9.32-kbp plasmid pTT8.The flagellin fliC gene(TTHC004)is encoded from plasmid pTT8.
The primers(Table1)were designed on the basis of conserved amino acid sequences of other flagellins from Thermotoga maritima,Bacillus sp.,Geobacillus sp., Salmonella and Clostridium sp.(Nelson et al.1999; Auvray et al.2001;Beatson et al.2006;Liu and Ochman 2007).Primers flag-1to flag-7were used as forward primers in all possible combinations with the reverse primers flag-8to flag-12.
The PCR conditions were as follows:denaturation at95°C for1min,annealing at70°C for1min and polymerization at 72°C for8min.These steps were repeated ten times and followed by three iterations of ten cycles,in which the anneal-ing temperature was68°C,63°C and60°C,respectively.A final elongation step was performed at72°C for20min.The resulting PCR products were analysed on1%(w/v)agarose gel and cloned into pGEM-T Easy vector(PROMEGA). JM109competent cells were transformed with the constructed plasmids,and positive candidates were sequenced with for-ward and reverse M13universal primers.LI-COR IR2DNA analyser and SequeTherm sequencing kit were used to se-quence the plasmid DNA.All primers,plasmids and strains used in this study are presented in Table1.
Dissociation of(FliC)1(FliS)2complex
To confirm the existence of the putative(FliC)1(FliS)2com-
plex,various treatments were carried out for its dissociation
such as heat treatment at various temperatures,acidic or
alkali treatment.Heat treatment was performed by maintain-
ing aliquots of isolated flagella at60°C,80°C and100°C.
After incubation,samples were cooled on ice and analysed
by western blot using anti-FliC.For acidic or alkali treat-
ment,100-μl aliquots of isolated flagella were diluted into
2ml of different solutions of pH2–11and incubated at65°
C for1h.For the different pH values,0.1M of the follow-
ing buffer solutions containing0.1%(v/v)Triton X-100
were used:KCl–HCl for pH2–3,CH3COONH4–CH3COOH for pH4.0–6.5,Tris–HCl for6.5–9.5,glycine–
NaOH for pH9.5–10.5and lysine–NaOH for pH10.5–11.
The suspensions were then centrifuged at20,000×g for
45min in order to separate the precipitated proteins of the
pellet from the supernatant.All fractions were analysed by
Western blotting using anti-FliC antibody.
Protein characterization:matrix-assisted laser
desorption–ionisation mass spectrometry
Protein sample was subjected to SDS–PAGE,and subse-
quently,the protein band was excised from the gel and
subjected to matrix-assisted laser desorption–ionisation
mass spectrometry(MALDI-MS)(Bruker Reflex III).
Following digestion with400ng trypsin,the resulting peptides were desalted using ZIP-TIPS(Millipore)and spot-ted onto Bruker gold anchor(AnchorChip400)targets with 2,5-dihydroxybenzoic acid matrix.Analysis was carried out in positive mode with a mass range of500–3,500Da.Data analysis was performed using Biotools software and Mascot search algorithms.
Results
Motility of T.thermophilus HB8and flagella formation Thermus bacteria cells are characterized as either immotile or motile(Huber and Stetter2004).To study the swimming and swarming motility of T.thermophilus HB8,plates com-posed of0.3%(w/v)and1.1%(w/v)agar in either rich medium(DSMZ-74)or in mineral salt medium containing 1.5%(w/v)sodium gluconate as carbon source were pre-pared.Inoculation with a small aliquot(2μl)of a fully grown culture of T.thermophilus was followed.
Our results revealed that T.thermophilus cells exhibited low swimming motility when grown for72h in DSMZ-74 agar0.3%(w/v)(Fig.1a)and much higher in MSM agar 0.3%(w/v)(Fig.1b).Although they were able to swim in low viscosity media,they did not show any swarming when transferred onto solid DSMZ-74or MSM media,even after 72h of cultivation(Fig.1c,d).
All filamentous structures present on T.thermophilus HB8membrane were isolated with an average yield of
Table1Primers,bacterial strains and plasmids used in this study Primer name Primer sequence Source or reference
flag-15′-GCCGGCGACGATGCGGCCGGT-3′This study
flag-25′-GGCTTGGCCATCTCCGAGAAG-3′This study
flag-35′-GGACTTGCAATAAGCGAGAAG-3′This study
flag-45′-GACGATGCCGCGGGCTTGGCGATC-3′This study
flag-55′-TTGATCCAGACCGCAGAGGGTGCA-3′This study
flag-65′-ACGGAGACCCACGCGATCTTGC AG-3′This study
flag-75′-ACCGAAACGCATGCAATCCTGCAT-3′This study
flag-85′-CAGGCATCGCAGGCTATGTTG-3′This study
flag-95′-CATGCAAACCAGCATCCACA TAAC-3′This study
flag-105′-CAGGCTAATCAGCAGCCTCAGAAC-3′This study
flag-115′-ATGTTGGCTCATGCCAATCAGCAG-3′This study
flag-125′-ATGCTGGCACAGG CGAACCAGCAT-3′This study
Strains Relevant characteristics Source or reference
T.thermophilus HB8Wild type DSM579(Deutsche
Sammlung Mikroogranismen) E.coli JM109recA1,endA1,gyrA96,thi,hsdR17,supE44,
relA1,Δ(lac-proAB)/F'[traD36,proAB+,
lacI q,lacZΔM15]
Promega
Plasmids Relevant characteristics Source or reference
pGEM-T Easy vector Linearized vector with single3′-T overhangs,
Amp r and blue/white selection
Promega
65μg/g of cells.Western blot analysis using anti-FliC was performed in the isolated filamentous structures from the ‘semisolid cultures ’which were grown in MSM or DSMZ-74(72h)and a band of molecular mass of 64kDa was immunodetected (Fig.2a ,lanes 1and 3).In contrast,flagella were not detected in the supernatants obtained from ‘solid cultures ’(Fig.2a ,lanes 2and 4).When T.thermophilus was grown in MSM liquid cultures,containing sodium gluconate as sole carbon source,flagellin was immunodetected after 24h in MSM and 36h in DSMZ-74of cultivation and was increased progressively at late stationary phase (Fig.2b ,c).Under these conditions,the presence of flagellin was also immunodetected intracellularly at 72h of cultivation,with a same molecular mass of 64kDa (Fig.2d ).The intracellular fraction was separated according to Auvray et al.(2001)while its purity was demonstrated by a negative immunos-taining with an antiserum raised against a periplasmic pro-tein (maltose-binding protein,New England Biolabs)(data not shown).A growth curve of T.thermophilus showing the time points of samples taken is presented in Fig.2e .
The protein profile of isolated filamentous structures of T.thermophilus obtained by SDS –PAGE revealed a major protein band of approximately 64kDa and a few faint
bands
Fig.1Swimming and swarming motility assay of T.thermophilus HB8.Swimming and swarming plates composed of 0.3%(w /v )(a ,b )and 1.1%(w /v )(c ,d )agar,in media:DSMZ-74(a ,c )and MSM (b ,d ),respectively,after 72h of
incubation
Fig.2a Western blot analysis of the isolated flagella from semisolid (lanes 1and 3)and solid (lanes 2and 4)media,using anti-FliC antibody.Either DSMZ-74(lanes 1and 2)or MSM (lanes 3and 4)medium was used.b Western blot analysis with anti-FliC antibody of the isolated flagella precipitated with TCA,from liquid MSM medium,containing 1.5%(w /v )sodium gluconate as carbon source and liquid DSMZ-74medium (c)at the following time intervals:5,18,24,36,48,60and 72h (lanes 1–7,respectively).d Western blot analysis of intracellular fractions with anti-FliC antibody at 72h of cultivation in MSM (1)or in DSMZ 74(2)medium.Molecular mass standards are indicated on the right.e Growth curve of T.thermophilus HB8in DSMZ-74medium (circle )and in MSM medium (square).Arrows indicate the time points of samples taken
(Fig.3a ).Only the 64-kDa protein was immunoreacted with the anti-FliC antibody (Fig.3b )indicating the existence of FliC and consequently,the formation of flagella filaments.When isolated flagella were electrophoresed under native conditions,a protein band of high molecular mass was obtained (data not shown).Since other filamentous struc-tures than flagella have been isolated in T.thermophilus HB27(Friedrich et al.2003),a closely relevant strain,a survey in T.thermophilus HB genome was performed for the putative pilin genes.Table 2summarizes T.thermophilus HB8putative genes encoding type IV pili and prepilin-like proteins.Based on the predicted molecular masses,the three faint bands observed in Fig.3a not immunodetected with anti-FliC could be attributed to pilin proteins.
Isolated flagella filamentous structures were also sub-jected to size exclusion chromatography (Fig.4),as de-scribed in “Materials and methods ”.Proteins of each fraction eluted from Sephadex G200were precipitated with TCA and analysed by SDS –PAGE on a 12%(w /v )gel.Samples were either boiled (for 30min)or not prior to electrophoresis.In the first case,immunostaining with
anti-FliC revealed a major band of approximately 28kDa in the primary fractions of the column (Fig.4,insert figure),where blue dextran (2,000kDa)was also eluted.Whereas in the second case,the protein band that was detected with anti-FliC in the same fractions presented a molecular mass of 64kDa (data not shown).These were the first indications that FliC of T.thermophilus forms a complex with another protein.The isolated 64-kDa protein complex was dissoci-ated by boiling for 30min and subjected to gel electropho-resis.As shown in Fig.5,it was clearly proven that FliC forms a complex with another protein of around 18kDa.As indicated from the size exclusion,chromatography flagellum has a molecular mass much higher than 200kDa.
The production of flagellum from T.thermophilus was further examined by immunofluorescence microscopy.
Cells
Fig.3Isolation of flagella from T.thermophilus cells grown in MSM containing 1.5%(w /v )sodium gluconate as sole carbon source.Coo-massie brilliant blue-stained SDS –PAGE (a )and western blot analysis with anti-FliC (Salmonella H antiserum)(b )of isolated https://www.doczj.com/doc/521823944.html,nes 1isolated flagella,M pre-stained molecular mass standards
Table 2Putative pilin genes of T.thermophilus HB8Gene Description
M W (kDa)TTHA0269Type IV prepilin peptidase,PilD 37.89TTHA1216Prepilin-like protein 16.94TTHA1217Prepilin-like protein 11.65TTHA1218Prepilin-like protein
13.12TTHA1221Pilin,type IV ,putative (putative PilA)12.86TTHA1222Pilin,type IV ,putative (putative PilA)13.18TTHA1774Pili retraction protein PilT 41.18TTHB117Putative type IV pilin 14.91TTHB119
Prepilin-like protein
33.54
Fig.5Thermal dissociation of the 64-kDa protein complex.Coomas-sie brilliant blue-stained SDS –PAGE (a )and western blot analysis with anti-FliC (Salmonella H antiserum)of the isolated flagella (lane 1)and the flagella after thermal treatment (lane 2
)
Fig.4Size exclusion chromatography of purified flagella from T.ther-mophilus cells.A Sephadex G200column (150×1cm)equilibrated with 50mM phosphate buffer,pH 7.0,was used.The molecular masses of the standards which are indicated with arrows are 1blue dextran 2,000kDa,2alcohol dehydrogenase 150kDa,3bovine serum albumin 67kDa,4ovalbumin 43kDa,5carbonic anhydrase 29kDa,6cytochrome c 12kDa and 7CrO 3-3kDa.Insert western blot analysis with anti-FliC of the fractions eluted from Sephadex G-200.The numbers of fractions are indicated above;line M pre-stained molecular mass standards
were grown in DSMZ-74(Fig.6a )and MSM medium supple-mented with sodium gluconate (Fig.6b –d )until log phase (Fig.6b ,d)and early stationary phase (Fig.6a ,c).Cells were fixed and flagellar filaments (FliC)were immunofluorescent-stained intensely green with FITC-conjugated to anti-FliC antibody which cross-reacted with FliC protein due to its abundance in filaments (Fig.6a –c ).A light image of cells grown in MSM until the early stationary phase is presented in Fig.6d .As it was observed,flagella were visible only when T.thermophilus was grown in MSM medium (Fig.6b ,c).The filament formation was more intense at the early stationary phase (Fig.6c ).In agreement with our immunoblotting experi-ments of the isolated shear fraction,the flagella filaments were not visible in a rich medium at the early stationary phase.
Identification of putative fliC encoding gene
To further support our findings,we proceeded with the iden-tification of the putative encoding gene in the genome of T.thermophilus HB8.Genomic DNA of T.thermophilus was provided by TAKARA.As described in “Materials and methods ”,12primers were used in various PCR amplifica-tions using genomic DNA of T.thermophilus as template.PCR products were cloned into pGEM-T-Easy vector,and their DNA sequence was analysed.One ORF,TTHC004,was identified (data not shown)that encodes an unknown protein of 27.7kDa.
The primary amino acid sequencing of TTHC004gene prod-uct is illustrated in Fig.7a .When blast analysis of the T.thermophilus TTHC004gene product was performed,similarity was detected with flagellin of several species (Fig.7a ).The highest degree of similarity was found with the flagellins of Salmonella enterica subsp.enterica serovar Typhi (gi 16503200;10.7%identity,16.9similarity),S.typhimurium LT2(gi 16765297;10.1%identity,17.9%similarity),T.mar-itima MSB8(gi 15643521;12.2%identity,19.3%similarity),Bacillus subtilis (gi 14278896;11.6%identity,19.1%similarity)and Bacillus licheniformis A (gi 52082071;13.9%identity,23.7%similarity).Interestingly,the highest similarity (34.3%)was detected among the N-terminal (4-140aa)of TTHC004gene product and the ‘N-terminal helical regions ’of flagellin proteins of other bacterial species (Fig.7a ,b).In addition,the region 192–249aa (C-terminal region)of the TTHC004gene product also exhibits 32.7%similarity with the ‘C-terminal helical region ’of other flagellins (Fig.7b ).The predicted mo-lecular mass of TTHC004gene product is in accordance with the band of 28kDa that was identified by anti-FliC immunostaining when samples were heated extensively prior to electrophoresis.
Identification of a (FliC)1(FliS)2complex
The detection with anti-FliC of a 64-kDa protein band when crude shear fractions of T.thermophilus HB8were not exten-sively boiled prior to electrophoresis led us to the
hypothesis
Fig.6Characterization of T.thermophilus flagella by immunofluorescence
microscopy.Cells were grown in DSMZ-74(a )and MSM medium supplemented with so-dium gluconate (b –d )until log phase (b ,d )and early stationary phase (a ,c ).Flagellar filaments (FliC)were stained green with FITC-conjugated anti-FliC an-tibody.Samples were visualized in a Leica SP confocal micro-scope equipped with a×100objective lens.A light image is also presented (d )
that a stable complex consisting of FliC and another protein could exist.Therefore,the isolated shear fractions were subjected to thermal treatment at various temperatures and time intervals.
atgggccaaaaccacgccatcggttgcggcccggtatccgtgctccggatcttggaagtc M G Q N H A I G C G P V S V L R I L E V taccgccagcggggcctggaggggattccctgggacatcaccctgaggctccccaaggac Y R Q R G L E G I P W D I T L R L P K D
gacctcagcttctcggggtactccgtcacggtgccggggagcgcaaggatcccctcaggt D L S F S G Y S V T V P G S A R I P S G
cttccccagtgggcccagctggccctctggccggtgaaactcaccacccagaacgggaag L P Q W A Q L A L W P V K L T T Q N G K
aacatctacaacgcctggatctcccaggccctgaacggcaagcaggctggactggaccag N I Y N A W I S Q A L N G K Q A G L D Q
gcggtcctgcccaaggactacgccccgggggctaaccgctggctggcggacaacggcctg A V L P K D Y A P G A N R W L A D N G L
ccggtgcgggtgacgggggcctacctgcgcgacttcgacgcctccttcctcctgaacccc P V R V T G A Y L R D F D A S F L L N P
tacatccccatcaccggccccatcgcctgggtgagctacacccagtacacctggaccacc Y I P I T G P I A W V S Y T Q Y T W T T
aacgggctgctcaaggacaccatcgggaatagggatcttccggcggtggttctctacgac N G L L K D T I G N R D L P A V V L Y D
agcgcacagatacgtagcgggtcgtcctactacctccactacgccccggcctaccgctac S A Q I R S G S S Y Y L H Y A P A Y R Y
cgggtggtggagtactgggactggtccgccaacttcgccacggtggacaacgggaaggag R V V E Y W D W S A N F A T V D N G K E
ggtctttccccccgtccttttggtcccattgagatttacctcagcgactactacgatctc G L S P R P F G P I E I Y L S D Y Y D L
tccaacggggtgtggcgcatcagccgctag S N G V W R I S R -
gi:15643521 MRINHNISALNAWRNISQTQYSMSKTLERLSSGLRINRAGDDAAGLAISEKMRGQIKGLN 60gi:52082071 MRINHNIAALNTLNRLSANNGASQKNMEKLSSGLRINRAGDDAAGLAISEKMRGQIRGLE 60TTHC004 MGQNHAIGCG-PVSVLRILEVYRQRGLEGIPWDITLRLPKDDLSFSGYSVTVPGSARIPS 59 * ** *.. . : : .: :* :. .: :. . ** : . * .: *. : .
gi: 15643521 MAIKNAQDAISLIQTAEGALTEVHSILQRMRELAVQAASD-TNTDVDREQIQKEIDQLRE 119gi:52082071 MASKNAQDGISLIQTAEGALTETHSILQRVRELVVQAGNTGTQQTEDLTAIQDEIKALKD 120TTHC004 GLPQWAQLALWPVKLTTQNGKNIYNAWISQALNGKQAGLDQAVLPKDYAPGANRWLADNG 119 : ** .: :: : .: :. **. : * .. .
gi: 15643521 EIDRIART--TEFNTKKLLDG------ 136 gi:52082071 EIDGISER--TEFNGKKLLDGTFK--- 124 TTHC004 LPVRVTGAYLRDFDASFLLNPYIPITG 146
:: :*: . **:
b
1 21 41 61 81 101 121 141 161 181 201 221 241 a
Fig.7a Identification of flagellin protein (FliC)as the product of TTHC004gene of T.thermophilus .The region 4–140aa (underlined peptides)revealed high similarity with the ‘N-terminal helical region ’of bacterial flagellins.The region 192–249aa (dashed underlined peptides)revealed similarity with the ‘C-terminal region ’of bacterial flagellins.b Multiple alignment of the N-terminal region (1–140aa)of TTHC004
gene product compared with the ‘N-terminal helical region ’of flagellin proteins of other species.Identical amino acid are marked with a star ;conserved amino acids are marked with double dot and semi-conserved amino acids are marked with single dot .NCBI accession numbers,given at the left ,refer to the following species:gi 15643521:T.maritima MSB8;gi 52082071:B.licheniformis ATCC 14580
Isolated flagellin complex retained its initial molecular mass of 64kDa even after 1h incubation at 60°C,or 80°C,while after treatment at 100°C for 20min,the initial flagellin complex was no longer detectable and a complex of ~46kDa was observed (Fig.8,lane 2).The stability and consequently the immunoreactivity of T.thermophilus fla-gellin complex were also affected at various pH values.At pH values ranging from 5to 8,flagellin retained its initial molecular mass,while at pH between 3to 4,the initial flagellin complex of 64kDa disappeared and the monomeric form of FliC of approximately 28kDa was detected (Fig.8,lane 3).At extreme pH values of 1–2or 9–11,neither the initial complex of flagellin nor any of the monomeric forms were immunodetected.Based on the resultant molecular mass of the complexes after each treatment and the predicted mass of 28kDa of TTHC004gene product,we assumed that FliC forms a stable complex with a homodimer of another protein of 18kDa.
The existence and the dissociation of this complex was also proved by dissolving the isolated shear fractions in acetate buffer pH 4.0and incubating for 2h at 65°C.SDS –PAGE analysis revealed that after the acetic treatment,the initial 64-kDa protein complex was disappeared and two
proteins bands of 28and 18kDa can be detected by Coomassie brilliant blue staining (Fig.9a ;lane 2).Only the protein of 28kDa was detected with anti-FliC antibody,as shown in Fig.9b .
The protein band of 18kDa (Fig.9a ;lane 2)was sub-jected to MALDI-MS and the amino acid sequences of the resulted peptides are presented in Fig.10.Blast analysis of these oligopeptides with T.thermophilus revealed that they matched completely with the protein encoded by TTHC003gene and has a molecular mass of 18.2kDa.The primary amino acid sequencing of TTHC003gene product is illus-trated in Fig.10a .When blast analysis of the T.thermophi-lus TTHC003gene product was performed,significant similarity was detected with chaperone FliS of S.typhimu-rium LT2(gi 16765299;13.1%identity,24.8%similarity),FliS of S.enterica subsp.enterica serovar Typhi (gi 16503202;13.1%identity,24.8%similarity)and FliS of P .aeruginosa (gi 7081486;10.6%identity,19.4%similarity)(Fig.10b ).These results indicated that TTHC003encodes the chaperon protein FliS of T.thermophilus and that FliC and FliS form a complex consisting of (FliC)1(FliS)2.Since FliS is a cytoplasmic protein,the identification of the (FliC)1(FliS)2in the isolated shear fraction can be justified by cell lysis due to the extensively shearing of bacterial cells that were in the late stationary phase.
Glycosylation and deglycosylation of T.thermophilus flagella
Purified flagella were analysed following SDS –PAGE with a fluorescent glycoprotein detection kit.The purified flagella reacted strongly in this assay,confirming the presence of carbo-hydrate molecules (Fig.11a ,lane 2).Glycosylation was also detected in cell-free supernatant (Fig.11a ,lane 1).Deglycosylation with N -glycosidase F shows a reduction in the molecular mass of the native form from 64to ~60kDa (Fig.11b ).Figure 11c reveals that deglycosylation did not affect the immunoreactivity of flagellin,since the resulted complex of ~60kDa was immunocross-reacted with anti-flagellin
antibody.
Fig.8Dissociation of (FliC)1(FliS)2complex of T.thermophilus .West-ern blot analysis of flagellin fragments,using anti -FliC (Salmonella H antiserum).Lanes :1(FliC)1(FliS)2complex without treatment;2(FliC)1(FliS)1complex after incubation at 100°C for 20min;3FliC monomeric form after incubation in 50mM CH 3COONH 4–CH 3COOH buffer,pH 4,for 1h at 70
°C
Fig.9Dissociation of (FliC)1(FliS)2complex by acetic treatment.Purified flagella were dissolved in acetate buffer pH 4.0and incubated for 2h at 65°C.Coomassie brilliant blue-stained SDS –PAGE (a )and western blot analysis with anti-FliC (Salmonella H antiserum)(b ).Lanes :1isolated flagella,2flagella after acetic treatment,3pre-stained molecular mass standards
Discussion
In the present study,questions concerning the motility and the presence of flagella in T.thermophilus as well as whether flagellum formation is depended on the environmental or nutritional conditions were approached.T.thermophilus is a swimming and not swarming motile bacterium,when it is cultured on solid media.Similar behaviour has been observed for other flagella-motile bacteria (Calvio et al.2005).Concerning the swimming ability of T.thermophilus ,our experiments demonstrated that motility was enhanced in pro-longed cultivation time,either in rich or mineral media.Under these conditions,phosphate was depleted as presented by Pantazaki et al.(2011).In the same study,glucose concentra-tion was also monitored during bacterial growth and was drastically reduced after 24h of cultivation.Therefore,it can be suggested that T.thermophilus initially remains non-motile in rich medium.Subsequently,when cells become more crowded or nutrients are depleted,they migrate rapidly from the point of inoculation to higher levels of nutrients.Our experiments revealed that T.thermophilus motility was ob-served earlier when cells were grown in mineral medium and it was achieved by producing the necessary proteins for fla-gella formation.The production of proteins necessary for
atgtactcctcggccttcaaggagagctccctctccgccaccctcgctgcctcgccgggg M Y S S A F K E S S L S A T L A A S P G tcctcaggaaggtccacgggctcatgggaggcgatgcgggaagcccaccggaagaacggc S S G R S T G S W E A M R E A H R K N G
ttcttggacgaactggcaaagcggtgggaccgactgattgagaagtcgcccccggtcccc F L D E L A K R W D R L I E K S P P V P
tctccccaggggctttctccccagtccctgggcggggatcccaagcccctggacctcgcc S P Q G L S P Q S L G G D P K P L D L A
acggagttcttccgggaccaggtgagcccacaggccacccttccgggacagtgcctctcc T E F F R D Q V S P Q A T L P G Q C L S
tggttcctgtgggcgtgcaccgcctaccggaacgcggggtacgtgtcttttaccgccagc W F L W A C T A Y R N A G Y V S F T A S
acgaacactttgccaggcgacttcgggaaccagcaaaacatttcgtacccttggtattgg T N T L P G D F G N Q Q N I S Y P W Y W
agaggaactcctaagcaggtctacaccaaccccctcaacgccccttccagagcgacccca R G T P K Q V Y T N P L N A P S R A T P
acaacacctggggagcctgggacctgaatgggccaaaaccacgccatcggttgcggcccg T T P G E P G T -
gi:16765299 MYTASGIKAYAQVSVESAVMSAS-PHQLIEMLFDGANSALVRARLFLEQGDVVAKGEALS 59 gi:16503202 MYTASGIKAYAQVSVESAVMSAS-PHQLIEMLFDGANSALVRARLFLEQGDVVAKGEALS 59 TTHC003 MYSSAFKESSLSATLAASPGSSGRSTGSWEAMREAHRKNGFLDELAKRWDRLIEKSPPVP 60 **::: :: ..:: :: *:. . * : :. .. . .* . . :: *. .:. gi:16765299 KAINIIDNGLKAG-----LDQEKGGEIATNLSELYDYMIRRLLQAN----------LRND 104gi:16503202 KAINIIDNGLKAG-----LDQEKGGEIATNLSELYDYMIRRLLQAN----------LRND 104TTHC003 SPQGLSPQSLGGDPKPLDLATEFFRDQVSPQATLPGQCLSWFLWACTAYRNAGYVSFTAS 120 . .: :.* .. * * : .: : * . : :* * : . gi:16765299 AQAIEEVEGLLSNIAEAW------KQISPKASFQESR----------- 135 gi:16503202 AQAIEEVERLLSNIAEAW------KQISPKASFQESR----------- 135 TTHC003 TNTLPGDFGNQQNISYPWYWRGTPKQVYTNPLNAPSRATPTTPGEPGT 168 :::: .**: .* **: .:. **
1 21 41 61 81 101 121 141 161 a
b
Fig.10a Assignment map of TTHC003gene product of T.thermo-philus .Boldface letters indicate peptides identified by MALDI-TOF-MS.b Multiple alignment of the TTHC003gene product of T.thermo-philus HB8compared with FliS proteins of other species.Identical amino acid are marked with a star ;conserved amino acids are marked
with double dot and semi-conserved amino acids are marked with single dot .NCBI accession numbers,given at the left ,refer to the following species:gi 16765299,Salmonella typhimurium LT2and gi 16503202,S.enterica subsp.enterica serovar Typhi
chemotaxis and therefore for flagella formation requires high energy (Larsen et al.1974;Adler 1975;Armitage 1992).It has been reported that carbon limitation in mineral medium in-duced chemotaxis in B.subtilis ,which is indicated by the strong induction of fliC and other flagellin-specific genes involved in chemotaxis and motility (Caramori and Galizzi 1998).Furthermore,flagellin levels have been reported to be highly induced,mainly at the end of growth in minimal media (Mirel and Chamberlin 1989).These enhanced levels of fla-gellin expression could be due to the parallel increase of carbon limitation in media containing,ammonia as a supple-mentary nitrogen source (Jurgen et al.2005).Further,experi-ments are in progress to verify the regulation of flagella formation in T.thermophilus .
Ralstonia eutropha H16is strongly flagellated in the exponential growth phase and loses a certain number of flagella when reach the stationary phase.Flagellation changes according to nutrient supply and state of poly (3-hydroxybutyrate)(PHB)accumulation (Raberg et al.2008).In the stationary phase,under conditions permis-sive for PHB biosynthesis,flagellation of cells admit-tedly stagnated.However,under conditions permissive for intracellular PHB mobilization,after the addition of a nitrogen source to carbon deprived cells,flagella are lost and this might be due to a degradation of flagella.In contrast,under nutrient limitation,cells retained their flagella (Raberg et al.2008).Our results are in accordance with these findings since conditions that enhanced flagella formation in T.thermophilus are also permissive for
polyhydroxyalkanoates biosynthesis as previously reported (Pantazaki et al.2003).
Flagellin in T.thermophilus was also detected intracellu-larly,as was expected,since synthesis of flagella proteins occurs in the cytoplasm in the form of soluble monomers (Ozin et al.2003).Flagellin binds to the low molecular mass (12kDa)chaperone FliS,which acts as a ‘bodyguard ’for FliC,preventing premature folding or inappropriate associa-tion of newly synthesized flagellin subunit in the cytosol by binding to their C-terminal interactive region prior to translo-cation (Auvray et al.2001;Ozin et al.2003).In Salmonella tryphimurium ,FliS forms homodimer,which with the contri-bution of the FliC monomer participate in the in vitro assem-bling of the complex (Auvray et al.2001).The 2:1stoichiometry was further indicated by the requirement for a 2:1FliS-to-FliC molar ratio for complete inhibition of FliC polymerization (Auvray et al.2001).The T.thermophilus (FliC)1(FliS)2complex of 64kDa remained intact under SDS –PAGE denaturating conditions similarly to the complex formed by S.tryphimurium (Auvray et al.2001);it is converted to a 46-kDa complex after heating at 100°C (FliC)1(FliS)1and to 28kDa (FliC)1monomer at acidic conditions.FliS of T.thermophilus was further determined by MALDI-MS.Although the fate of FliS after FliC entry into the flagella pore has not yet well been understood,the detection of (FliC)1(FliS)2complex in the extracellular medium or in the shear fractions of T.thermophilus could be attributed to cell lysis.
Flagellin was also reported to be unfolded or denatured under heat (Fedorov and Kostyukova 1984;V onderviszt et al.1990).The stability of flagellin is also influenced by changes in pH (Klein et al.1968;Aizawa 1996).T.thermophilus flagellin in the extracellular medium retained both its molec-ular mass and antigenic properties when the pH ranged be-tween 5and 8,while detection of FliC was no longer possible in acetic conditions (data obtained but not shown).
It is worth noticed that during the isolation process of flagella from T.thermophilus HB8,other filamentous struc-tures were also obtained.A survey in T.thermophilus HB8genome revealed a significant number of putative pilin or pilin-like proteins.Therefore,it is possible some of them to be co-isolated with flagella.Such structures have already been detected in the closely related strain T.thermophilus HB27and were implicated in the competence for natural transformation and type IV pilus biogenesis (Friedrich et al.2003).
T.thermophilus flagellin is glycosylated similarly to flag-ellins from other species (for a review,see Logan 2006).N -Glycosylated flagellins have also been detected in Serpulina hyodysenteriae (Li et al.1993),Borrelia burgdorferi (Ge et al.1998),Halobacterium salinarum (Wieland et al.1985)and Methanococcus voltae (Voisin et al.2005).Treatment of T.thermophilus flagellin with N -glycosidase F resulted in a 60-kDa protein which was also detectable with anti-FliC ,revealing that deglycosylation did not influence
the
Fig.11Glycan staining and deglycosylation of T.thermophilus flagella.a Staining of glycosylated proteins with fluorescent glycoprotein detec-tion https://www.doczj.com/doc/521823944.html,ne 1cell-free https://www.doczj.com/doc/521823944.html,ne 2purified flagella from sodium gluconate-grown cells.b SDS –PAGE and silver staining of native (lane 1)and deglycosylated flagella after treatment with N -glycosidase F (lane 2).c Western blot analysis with anti-FliC (Salmonella H antiserum).Lanes as indicated in legend (b ).Molecular mass standards are indicated on the right
antigenic properties of flagellin.Hence,it can be assumed that glycosylation probably protects the flagellin protein at high temperatures similarly to other thermophilic proteins. Flagellin glycosylation was also identified in thermophilic Bacillus species,and it was speculated that glycosylation is required for flagellar filament assembly of these bacilli (Hayakawa et al.2009).The importance of flagellin glyco-sylation has been also reported for motility and virulence (Goon et al.2003;Arora et al.2005;Taguchi et al.2008). Finally,the low molecular mass of T.thermophilus flagellin monomer(28kDa)is in accordance to Geobacillus kausto-philus flagellin that was reported to possess a molecular mass of29.3kDa(Hayakawa et al.2009).
Even though the entire genome of T.thermophilus HB8 has been identified,many hypothetical proteins are not yet characterized(Lioliou et al.2004).The possible functions of two thirds of putative proteins were based on the sequence homology to proteins from other genomes and not on ex-perimental evidence(Kondo et al.2004).Our results indi-cated the existence of flagellin in T.thermophilus and the gene locus that encodes flagellin,TTHC004,which is locat-ed in the second megaplasmid of T.thermophilus,pTT8.T. thermophilus flagellin revealed considerable similarity (16.9–23.7%)with the flagellin protein of T.maritima and several strains of Salmonella and Bacillus species. TTHC003,which is close to TTHC004,encodes a FliS protein of18kDa,while blast analysis of TTHC003 revealed19.4%up to24.8%similarity with FliS proteins of several Salmonella and Pseudomonas species.
Our finding that T.thermophilus is able to possess flagella filaments arise new perspectives in research,since thermostable flagellins may have interesting applications in biotechnology and in nanotechnology(Malapaka and Tripp2006).Flagellin filaments from T.thermophilus could be excellent candidates for the development of novel hybrid nanostructures,or nano-tubes.They could be used also for the extracellular display of thermostable proteins and peptides with sensor,or catalytic activity,as it was suggested by Martin and Kohli(2003). References
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