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Expression of the htrB gene is essential for responsiveness of Salmonella typhimurium and Campylobacter jejuni to harsh environments

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Expression of the htrB gene is essential for responsiveness of Salmonella typhimurium and Campylobacter jejuni to harsh environments
  Downloaded from byIP: Thu, 22 Dec 2016 23:20:52 Expression of the  htrB  gene is essential forresponsiveness of  Salmonella typhimurium  and Campylobacter jejuni   to harsh environments Vongsavanh Phongsisay, Viraj N. Perera and Benjamin N. Fry Correspondence Benjamin N. School of Applied Sciences, Royal Melbourne Institute of Technology University, Bundoora,Melbourne, Victoria 3083, Australia Received  19 June 2006 Revised  5 September 2006 Accepted  18 September 2006 In  Campylobacter jejuni  , an  htrB  homologous gene is located in the lipo-oligosaccharide synthesisgene cluster. This study examined the effects of  htrB  expression on the responsiveness of Salmonella typhimurium  and  C. jejuni   to harsh environments. Complementation experimentsshowed that the  C. jejuni htrB  gene could restore the normal morphology of the  Salmonella htrB mutant, and its ability to grow without inhibition under heat, acid and osmotic stresses, but not bilestress. This indicated that the  htrB  genes in  C. jejuni   and  S. typhimurium  exhibit similarpleiotropiceffects.Moreover,quantitativereal-timeRT-PCRshowedthatexpressionofthe C.jejuni htrB  gene was upregulated under acid, heat, oxidative and osmotic stresses, but did not changeunder bile stress. This indicated that the  C. jejuni htrB  gene plays a role in regulating cellresponses to various environmental changes. Furthermore, deletion mutation of the  htrB  gene in  C. jejuni   was lethal, indicating that the  htrB  gene is essential for  C. jejuni   survival. Therefore, theseresultsshowedthatexpressionofthe htrB geneisessentialfortheresponseof S. typhimurium and C. jejuni   to environmental stresses. INTRODUCTION Modulation of lipid A acylation in Gram-negative bacteriaresults in pleiotropic effects. In  Escherichia coli  , mutation of the  htrB   gene encoding a lipid A acyltransferase leads toinhibitionofbacterialgrowth athigh temperature(Karow& Georgopoulos, 1991; Karow   et al. , 1991), morphologicalchange from short to filamentous rods (Karow   et al. , 1991),and unusually increased bile resistance (Karow & Georgopoulos, 1992). In  Salmonella typhimurium , inactiva-tion of the  htrB   homologous gene exhibits notonly the sameeffects as those seen in  E. coli  , but also hyperflagellation andseverely limited virulence (Jones  et al. , 1997; Sunshine  et al. ,1997). In  Haemophilus influenzae  ,knockout of the  htrB  generesults in increased bile sensitivity (Lee  et al. , 1995),increased sensitivity to human antimicrobial peptides ( b -defensins) (Starner  et al. , 2002), decreased colonizationcapacity (Swords  et al. , 2002), decreased intracellularviability (Swords  et al. , 2002), and decreased pro-inflam-matory cytokine induction (Tong  et al. , 2001), but it doesnot affect morphology (Lee  et al. , 1995). Campylobacter jejuni   is an enteric bacterium causing humangastroenteritis worldwide (Coker  et al. , 2002; O’Ryan  et al. ,2005). The predominant symptoms are inflammatory diarrhoea, abdominal pain, and/or fever. Poultry products,milk and water are frequently reported as the infectioussources. Infections are mainly observed in children under5 years of age, and cause serious complications inimmunocompromised hosts (Monselise  et al. , 2004;Ramon Maestre  et al. , 2001).The lipo-oligosaccharide (LOS) of   C. jejuni   is a majorsurface molecule consisting of two parts, the coreoligosaccharide and lipid A. The core region is involvedin virulence (Fry   et al. , 2000) and induction of Guillain-Barre´ syndrome, an autoimmune neuropathy of theperipheral nervous system (Yuki  et al. , 2004). The lipid Aof the LOS molecule possesses endotoxic properties (Naess& Hofstad, 1984).This crucial molecule is partly encoded by the  wla  II gene cluster, which shows a high degree of variation among strains (Gilbert  et al. , 2002; Parker  et al. ,2005). In  C. jejuni   strain HB 93-13, it contains 13consecutive genes:  waaC  ,  htrB  ,  wlaNC  ,  wlaND  ,  cgtA ,  cgtB  , cstII  ,  neuB  ,  neuC  ,  neuA ,  wlaVA ,  wlaQA  and  waaF   (GenBank accessionno. AY297047).The  htrB  homologue foundinthis wla  II gene cluster is conserved in  C. jejuni  , and is similar tothe  htrB   gene of   S. typhimurium ,  E. coli   and  H. influenzae  .Functionally, the  C. jejuni htrB   gene encodes a putativeacyltransferase involved in lipid A synthesis (Gilbert  et al. ,2000, 2002; Parkhill  et al. , 2000). This study examined theeffects of   htrB   expression on the responsiveness of   S.typhimurium  and  C. jejuni   to harsh environments,using complementation, gene expression and mutationexperiments. Abbreviations:  C T , cycle threshold; DOC, sodium deoxycholate; Km,kanamycin resistance cassette; LOS, lipo-oligosaccharide. 254 0002-9230  G  2007 SGM  Printed in Great BritainMicrobiology   (2007),  153 , 254–262  DOI  10.1099/mic.0.29230-0  Downloaded from byIP: Thu, 22 Dec 2016 23:20:52 METHODS Bacterial strains and growth conditions.  C. jejuni   strains HB93-13 (Ho  et al. , 1995), O:4, O:41, O:36, 81116 (Palmer  et al. ,1983), NCTC 11168, ATCC 43446 and OH 4382 (Aspinall  et al. ,1994) were included in this study.  C. jejuni   was grown on Columbiaagar plates supplemented with 5% (v/v) defribinated horse bloodunder microaerobic conditions (5% O 2 , 10% CO 2 , 85% N 2 ) at42 u C for 16 h, unless otherwise stated.  E. coli   DH5 a  was grown inLuria–Bertani (LB) broth or agar at 37 u C for 16 h.  S. typhimurium strains SL1344 (wild-type) and SL1344  htrB1 :: Tn  10 ( htrB   mutant,tetracycline resistance) were kindly provided by Dr B. D. Jones,Department of Microbiology, University of Iowa (Jones  et al. , 1997;Sunshine  et al. , 1997).  S. typhimurium  was grown in LB broth or onLB agar at 30 u C for 16 h, unless otherwise stated. Media were sup-plemented with 150  m g ampicillin ml 2 1 , 15 or 50  m g kanamycinml 2 1 , 20  m g tetracycline ml 2 1 , 2% (w/v) X-Gal in dimethyl forma-mide (40  m l for each LB plate), and 100 mM IPTG (40  m l for eachLB plate), when appropriate. Analysis of DNA and amino acid sequences.  Clone managerversion 6 (Scientific and Education Software) was used to designprimers, plan cloning and analyse DNA and amino acid sequences.Primers were designed using the  wla  II–LOS synthesis gene cluster of  C. jejuni   strain HB 93-13 (GenBank accession no. AY297047), unlessotherwise stated. DNA manipulation.  Plasmid DNA was isolated using the mini-prepprocedure described by Ausubel  et al.  (1995), and/or the QIAprepSpin Miniprep kit (Qiagen) according to the manufacturer’s instruc-tions. The DNA quantity was determined using spectrophotometry.Restriction enzymes, T4 DNA ligase and alkaline phosphatase werepurchased from Promega, and used according to the manufacturer’sinstructions. Restriction mapping was performed to confirm thecomposition and size of the constructed plasmids by digestion withappropriate restriction enzymes. Transformation of   C. jejuni   withplasmid or genomic DNA was performed using electroporation(25  m F, 1.25 kV and 600  V , gene pulser apparatus; Bio-Rad) and/ornatural transformation (biphasic technique), as described by Wassenaar  et al.  (1993). Transformation of   E. coli   and  S. typhimur-ium  with plasmid DNA was performed using electroporation (25  m F,2.48 kV and 200  V , gene pulser apparatus; Bio-Rad). Competent cellsfor  E. coli   and  S. typhimurium  were prepared in cold 10% (v/v) gly-cerol as described by Sambrook & Russell (2000).  pfu -PCR.  pfu  -PCR was used to amplify a DNA fragment from puri-fied chromosomal DNA. The reaction mixture was prepared in a50  m l total volume of 1 6  pfu   buffer containing 200  m M each of dATP, dTTP, dCTP and dGTP, 100 ng of each primer, 100 ngDNA, and 5 U  pfu   polymerase (Roche). The PCR conditions were asfollows: 94 u C for 3 min (initial denaturation); 35 cycles of 94 u C for30 s (denaturation), 50 u C for 1 min (annealing), 72 u C for  x   min(extension); and 72 u C for 7 min (final elongation).  x   was calculatedby dividing the length of the PCR product by 500 bp, as  pfu   poly-merase synthesizes 500 bp min 2 1 . Colony-PCR.  Colony-PCR was used to screen transformants carry-ing new constructs. The reaction mixture was prepared in a 50  m ltotal volume of 1 6 Taq   buffer containing 1.5 mM MgCl 2 , 200  m Meach of dATP, dTTP, dCTP and dGTP, 100 ng of each primer, and2.5 U  Taq   polymerase (ABI). A 200  m l (maximum volume) tip wasused to gently touch a colony on a culture plate, and the colony material was directly mixed into a PCR tube containing masterreagent, which had been prepared beforehand. The PCR conditionswere as follows: 94 u C for 10 min; 35 cycles of 94 u C for 30 s, 50 u Cfor 1 min, 72 u C for  y   min; and 72 u C for 7 min.  y   was calculated by dividing the length of the PCR product by 1000 bp, as  Taq   polymer-ase synthesizes 1000 bp min 2 1 . Construction of a plasmid carrying the  C. jejuni htrB  genein the  S. typhimurium htrB  mutant.  A 917 bp DNA fragmentcontaining 18 bp of the  waaC   gene (upstream adjacent gene), theentire  htrB   gene (888 bp) and 11 bp of the  wlaNC   gene (down-stream adjacent gene) of   C. jejuni   strain HB 93-13 was amplified by   pfu  -PCR with primers  Bam HI-waaC-F (5 9 -TTGCCAAAGGATCCC-TTAATGAAAAATAGTGATAG-3 9 ) and  Cla  I-wlaNC-R (5 9 -TTGT-TATCGATTCATTTTGCACCCTTGT-3 9 ). A PCR product wascloned into the pBluescript plasmid in the same orientation as theampicillin-resistance cassette, using the  Bam HI and  Cla  I sites, andthe resultant construct was subsequently introduced into  E. coli  DH5 a  by electroporation. Transformants carrying the  htrB   gene con-structs were screened using colony-PCR with primers 172-pBlue-F(5 9 -GGTTCCGATTTAGTGCTTTA-3 9 ) and 825-pBu-R (5 9 -GAA-ACAGCTATGACCATGAT-3 9 ). These primers were designed toamplify a 1516 bp plasmid fragment, which included a 917 bpinserted PCR product. The pBluescript carrying the  C. jejuni htrB  gene (named pBlue  htrB  + ) was isolated from  E. coli  , and thenintroduced into the  S. typhimurium htrB   mutant by electroporation. Examination of bacterial growth and morphology.  The wild-type, mutant and complemented  Salmonella   strains were grown onLB agar plates at 30, 37 and 42 u C for 24 h to OD 600  0.3. The culturemedia were supplemented with appropriate antibiotics. Tetracyclinewas added to the growth medium used for the  S. typhimurium mutant, while ampicillin and tetracycline were added to the growthmedium used for the complemented strain. The ability of bacteria togrow at 30, 37 and 42 u C was observed. A Gram stain was performedand the morphology was observed under a light microscope. Examination of bacterial sensitivity to acid and osmotic stresses.  To test for acid sensitivity, the wild-type, mutant andcomplemented  Salmonella   strains (OD 600  0.3) were grown at 30 u Cin 0.1% (w/v) peptone water, pH 2.5–7.0, for 24 h. After incuba-tion, cell density was measured at OD 600 , and an equal volume of culture medium was distributed on LB agar plates. All culture mediawere supplemented with appropriate antibiotics as described above.The plates were incubated at 30 u C for another 24 h. To test forosmotic sensitivity, the bacteria were grown as described above,except that 0.1% peptone water (pH 7.0) was supplemented withNaCl (1–10%, w/v). Examination of bacterial sensitivity to bile stress.  To test forbile sensitivity, the wild-type, mutant and complemented  Salmonella  strains (OD 600  0.3) were grown on LB agar plates containing sodiumdeoxycholate (DOC; 2, 4, 6, 8 and 10%). The culture media weresupplemented with appropriate antibiotics as described above. Theplates were incubated at 30 u C for 24 h. Treatment of  C. jejuni   with stress environments, and RNApreparation.  Prior to the treatment of   C. jejuni   with stress environ-ments, the ability of   C. jejuni   HB 93-13 to grow in brucella brothsunder heat, acid, osmotic, oxidative or bile stresses was examined. Agrowth temperature of 44 u C was selected as heat stress, since  C. jejuni   was able to grow at 43 u C but not at 45 u C. pH 5.5 wasselected as acid stress, since  C. jejuni   showed normal growth atpH 6.0, inhibited growth at pH 5.5, and no growth at pH 5.0. ANaCl concentration of 1.5% was selected as osmotic stress, since thebacteria showed normal growth at 1% NaCl, inhibited growth at1.5% NaCl, and no growth at 2% NaCl. As  C. jejuni   was unable togrow in normal atmospheric conditions, these were used for the oxi-dative stress challenge. For bile stress, 500  m g DOC ml 2 1 was used(Lin  et al. , 2005). C. jejuni   HB 93-13 was grown in 30 ml brucella broth with gentleshaking under microaerobic conditions at 37 u C for 19 h, and 100  m lbacterial culture was aliquoted into six bottles of brucella broth(30 ml), and incubated for another 19 h. After incubation, 1 ml 255 htrB   and  C. jejuni   response to harsh environments  Downloaded from byIP: Thu, 22 Dec 2016 23:20:52 brucella broth, which had been supplemented with concentrated HCl,NaCl or DOC, and pre-warmed at 37 u C, was added to the culturebottles to obtain a final pH of 5.5 (acid stress), a NaCl concentration of 1.5% (osmotic stress), and a DOC concentration of 500  m g ml 2 1 (bilestress). The fourth culture bottle, with 1 ml pre-warmed brucella brothadded, was used as the calibrator (normal  htrB   expression level). Thefifthculturebottlewasimmediatelymovedtothe44 u Cincubator(heatstress). Incubation was performed at 37 u C, except for the fifth culturebottle, with gentle shaking under microaerobic conditions. Thebacteria from the sixth culture bottle were poured onto cultureplates, and incubated at 37 u C with gentle shaking under normalatmospheric conditions (oxidative stress). Culture samples werecollected after incubation for 15 and 30 min, and transferred directly into a 1/10 volume of cold 10 6 stop solution  [  5% (v/v) phenol in100% ethanol ]  to halt transcription and RNA degradation. RNAsamples were isolated using the RNAgents Total RNA Isolation system(Promega), and DNA decontamination was performed using theTURBO DNA-  free   kit (Ambion). Quantitative real-time RT-PCR.  Primers used for cDNA synthesiswere as follows. The primers q-htrB-R (5 9 -TTGAGTGTATTGAG-GAAAAC-3 9 ), q-16S rRNA-R (5 9 -GTATTCTTGGTGATATCTAC-3 9 ;accession no. AL111168), q-luxS-R (5 9 -ATAAATCCTGCGAATA-AATG-3 9 ; accession no. AL111168) and q-rpoA-R (5 9 -ATTT-GTCCATCAGTTGTTAC-3 9 ; accession no. AL111168) were used forsynthesis of cDNA for the  htrB  , 16S rRNA,  luxS   and  rpoA genes  ,respectively.cDNA synthesis was performed using the ImProm-II reversetranscriptase (Promega). One microgram of RNA and 50 ng anti-sense primer in a total volume of 5  m l were heated at 70 u C for 5 min,and immediately chilled on ice for at least 5 min. The master mix wasprepared in a total volume of 15  m l, which consisted of 5  m l RNase-freewater, 4  m l 5 6 ImProm-II reaction buffer, 2  m l MgCl 2  (25 mM), 1  m ldNTP mix (10 mM each dNTP), 2  m l recombinant RNasin ribonu-clease inhibitor (5 U  m l 2 1 ), and 1  m l Improm-II reverse transcriptase(1  m l per reaction). The master mix was dispensed into the reactiontube containing the mixture of heated RNA and primer. The tubewas gently mixed, followed by incubation at 25 u C for 5 min, 42 u Cfor60 min,and70 u Cfor15 min.Reversetranscriptasewassubstitutedby RNase-free water for the negative control. After cDNA synthesis,10  m l RNase A solution (20  m g ml 2 1 ) was added to the reactionmixture, incubated at 37 u C for 20 min, and 220  m l water wasadded. For each gene, a dilution series of newly synthesized cDNAwas made and included in a quantitative PCR to examine the efficiency of PCR.Primers used for PCR were as follows. The primers q-htrB-F (5 9 -TTATGCCTGATTGTATCTTG-3 9 ) and q-htrB-R, as described above,were used to amplify a 125 bp fragment of the  htrB   gene-specificcDNA. The primers q-16S rRNA-F (5 9 -GTCTCTTGTGAAATCT-AATG-3 9 , accession no. CJ11168X3) and q-16S rRNA-R, as describedabove, were used to amplify a 123 bp fragment of 16S rRNA gene-specific cDNA. The primers q-luxS-F (5 9 -AAGTTATGAAAACACC-TAAG-3 9 , accession no. CJ11168X4) and q-luxS-R, as described above,were used to amplify a 124 bp fragment of   luxS   gene-specific cDNA.The primers q-rpoA-F (5 9 -GCTTTAGATGCTTTCTTTAC-3 9 , acces-sion no. CJ11168X6) and q-rpoA-R, as described above, were used toamplify a 119 bp fragment of the  rpoA  gene-specific cDNA.A quantitative PCR was performed on the MyiQ PCR detection system(Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad), according to themanufacturer’s instructions. The PCR reaction was performed in atotal volume of 25  m l, which contained 12.5  m l iQ SYBR GreenSupermix and 12.5  m l master mix, consisting of 1  m l forward primer(50 ng  m l 2 1 ),1  m lreverseprimer(50 ng  m l 2 1 ),3  m ldilutedcDNA,and7.5  m l water. Each PCR was performed in duplicate. The same PCR samples that showed variant values of the cycle threshold (C T ) of morethan 1 were repeated. PCR conditions were as follows: cycle 1 ( 6 1),95 u C for 5 min; cycle 2 ( 6 35), 95 u C for 30 s, 55 or 60 u C for 30 s(55 u C for  luxS  ; 60 u C for  htrB  , 16S rRNA gene and  rpoA ), 72 u C for30 s; cycle 3 ( 6 100), 95 u C for 10 s (decrease setpoint temperatureafter cycle 2 by 0.5 u C, enabling melt-curve data collection andanalysis). Each specific amplicon was verified by the presence of both asinglemelting-temperature peakandasinglebandofexpectedsizeona3% agarose gel after electrophoresis. C T  values were determined withthe MyiQ software (Bio-Rad). The relative changes ( x  -fold) in geneexpression between the induced and calibrator samples were calculatedusing the 2 2 DD CT method, as described by Livak and Schmittgen(2001).The16SrRNA, rpoA and/or luxS  geneswereusedastheinternalcontrols. A relative expression value of more than twofold wasconsidered as significant up- or down-regulation. Construction of the  C. jejuni htrB  mutant.  A pBluescript plas-mid carrying the mutated  htrB   gene of   C. jejuni   HB 93-13 was con-structed. Firstly, a 646 bp DNA fragment containing the partial waaC   and  htrB   genes of   C. jejuni   HB 93-13 (nt 140–785; accessionno. AY297047) was amplified by the  pfu  -PCR with primers  Eco  RI-waaC-F1 (5 9 -ATAGGAATTCATAGCGGTCCAACACA-3 9 ) and Bam HI-htrB-R1 (5 9 -AATCGGATCCTATTTAGCCGCATAAGC-3 9 ).The PCR product was cloned into pBluescript in the forward direc-tion via the  Eco  RI and  Bam HI sites. The resultant construct was sub-sequently introduced into  E. coli   DH5 a  by electroporation. Apositive clone was selected on LB agar supplemented with ampicillin,X-Gal and IPTG, according to the blue and white phenotypes. Thisprocedure identified the pBluescript carrying the partial  waaC   and htrB   genes (named pBluA). Secondly,  pfu  -PCR with primers Bam HI-htrB-F2 (5 9 -TTACGGATCCAGACTGCGTAGAAAACGA-3 9 ) and  Xba  I-wlaNC-R2 (5 9 -CCTTTCTAGAGATTTTTACGGCT-AAGTG-3 9 ) was used to amplify a 678 bp DNA fragment containingthe partial  htrB   and  wlaNC   genes (nt 934—1611; accession no.AY297047). This PCR product was cloned into pBluA in the forwardorientation via the  Bam HI and  Xba  I sites. Positive clones were iden-tified by colony hybridization using the second PCR product,labelled with DIG using the DIG labelling kit (Roche), as a probe.The colony-DNA probe hybridization was performed at 65 u C over-night, and detection was performed using the alkaline phosphate-conjugated anti-DIG antibody and the nitro-blue tetrazolium chlor-ide/5-bromo-4-chloro-3 9 -indolyphosphate  p  -toluidine salt (NBT/BCIP) substrate, as described in the user’s guide handbook (Roche).Positive clones carrying the  htrB   gene with a 150 bp (nt 785–934)deletion were named pBluB. A 1494 bp kanamycin resistance cas-sette (Km) was cloned into pBluB using the  Bam HI site. Thisresulted in a construct containing part of the  htrB   gene interruptedby the Km (named pBluC), in which the Km was flanked by a646 bp upstream and 678 bp downstream DNA fragments. ThepBluC carrying the Km in the same direction as the  htrB   gene wasnamed pBluCF, while the construct carrying the Km in the oppositedirection to the  htrB   gene was designated pBluCR. Finally, theseconstructed plasmids were confirmed by sequencing using theABI sequencing mix V3.1 (ABI), according to the manufacturer’sinstructions.Natural transformation and electroporation were used to introduceeach recombinant plasmid (pBluCF and pBluCR) into  C. jejuni   strainsHB 93-13, O:4, O:41, O:36, 81116, 11168, ATCC 43446 and OH4382. pBluescript plasmids carrying the Km within the  wlaVA  gene(pBlu11KR) or the  waaF   gene (pBlu13KF) were used as the positivecontrols. pBluescript alone was used as the negative control.Transformants were screened on 5% blood agar plates supplementedwith kanamycin (15  m g ml 2 1 ). Culture media were incubated undermicroaerobic conditions at 30, 37 and 42 u C for 5 days. 256  Microbiology   153 V. Phongsisay, V. N. Perera and B. N. Fry  Downloaded from byIP: Thu, 22 Dec 2016 23:20:52 RESULTS AND DISCUSSION Characteristics of the  C. jejuni htrB  gene The  htrB   homologue of   C. jejuni   HB 93-13 is located in the wla  II–LOS synthesis gene cluster. DNA sequence analysis of the C.jejunihtrB  geneshowedanORFof888 bp,startingwitha methionine when translated. Under microaerobic condi-tions, the  htrB   gene and the other LOS synthesis genes weretranscribed as part of several operons using multipletranscriptional start sites with promoters upstream of thestartcodonsofthe Cj1132c  , waaC  , cgtA , cgtB  , cstII  , wlaQA and waaF   genes (V. Phongsisay, unpublished data). Multiplesequencealignmentsshowedthatthe C.jejuni  HB93-13HtrBprotein was similar to  E. coli   HtrB (20%; accession no.NC_004431),  S. typhimurium  HtrB (20%; accession no.NC_003197) and  H. influenzae   HtrB (20%; accessionno. NC_000907) proteins. Among other bacteria,  S. typhi-murium HtrBshowed78%similarityto E.coli  HtrBand54%similarityto H.influenzae  HtrB.Theseresultsshowedthatthe S. typhimurium ,  E. coli   and  H. influenzae   HtrB proteins weremore closely related to each other than to  C. jejuni   HtrBprotein. In  S. typhimurium , mutation of the  htrB   gene, whichencodes an acyltransferase enzyme involved in lipid Asynthesis, results in pleiotropic effects in both the pathology and physiology of   S. typhimurium . These effects includemorphological changes from short to filamentous rods,hyperflagellation, inability to grow at high temperatures,increased bile resistance, and reduced virulence (Jones  et al. ,1997; Sunshine  et al. , 1997).In thisstudy, the  S. typhimuriumhtrB   mutant was used as a model for studying the pleiotropiceffectsresultingfromexpressionofthe C.jejunihtrB  gene.The S.typhimuriumhtrB  mutantwascomplementedwiththe htrB  gene from  C. jejuni   HB 93-13. The wild-type, mutant andcomplemented  Salmonella   strains were characterized. Role of the  C. jejuni htrB  gene in  S. typhimurium  morphology To examine whether expression of the  C. jejuni htrB   geneaffected the morphology of the  S. typhimurium htrB   mutant,the wild-type, mutant and complemented  Salmonella   strainsweregrownonLBagarplatesat30,37and42 u Cfor24 h.Theculture media were supplemented with appropriate antibio-tics. A Gram stain was performed and the morphology wasobserved under a light microscope. The morphology of thecomplemented and wild-type strains was similar, showingGram-negative short rods at 30, 37 and 42 u C, while themorphologyofthemutantdisplayedGram-negative,filamen-tous, bulging, short rods at all temperatures tested (Fig. 1a).Theseresultsshowthatthe C.jejunihtrB  genecouldrestorethewild-typemorphologyofthe S.typhimuriumhtrB  mutant,andhence its expression affected the bacterial morphology. Role of the  C. jejuni htrB  gene in growth of  S. typhimurium  at high temperature To examine whether expression of the  C. jejuni htrB   geneaffectedthecapacityofbacteriatogrowathightemperature,thewild-type,mutantandcomplemented Salmonella  strainswere grown on LB agar plates, as previously described. Theresults showed that the  C. jejuni htrB   gene could restore theability of the  S. typhimurium htrB   mutant to grow at hightemperatures, as the complemented and wild-type strainsgrew normally at 30, 37 and 42 u C, while the mutant wastemperature-sensitive and showed inhibited growth at 37and 42 u C(Fig. 1b). Thisshows thatthe  C. jejuni htrB  geneisessential for the  S. typhimurium htrB   mutant to grow properly at high temperature. A previous study has shownthat the  S. typhimurium htrB   mutant is unable to grow at37 u C (Sunshine  et al. , 1997), while in this study, thisbacterial strain showed inhibited growth at 37 u C (Fig. 1b).These inconsistent results may be explained by a loss of temperature sensitivity of the  S. typhimurium htrB   mutantafter a few passages at 30 u C, which has also been observedfor the  H. influenzae htrB   mutant (Lee  et al. , 1995). Role of the  C. jejuni htrB  gene in acid sensitivity of  S. typhimurium To examine whether expression of the  C. jejuni htrB   geneaffected acid sensitivity, the wild-type, mutant andcomplemented  Salmonella   strains were grown at 30 u C in0.1% (w/v) peptone water (pH 2.5–7.0) for 24 h. Afterincubation, the cell density was measured at OD 600 , and anequal volume of culture medium was distributed on LB agarplates. All culture media were supplemented with appro-priate antibiotics, as described above. The plates wereincubated at 30 u C for another 24 h. The results showed thatthe mutant strain exhibited less growth on LB agar platescompared to the wild-type, and the complemented strainshowed growth intermediate between those of the wild-typeand mutant strains at lower pH (Fig. 1c, selected results of bacterial growth on LB plates). Therefore, expression of the C. jejuni htrB   gene contributed to the acid tolerance of the S. typhimurium htrB   mutant. Role of the  C. jejuni htrB  gene in high osmotic sensitivity of  S. typhimurium To examine whether expression of the  C. jejuni htrB   geneaffected osmotic sensitivity, the wild-type, mutant andcomplemented  Salmonella   strains were grown as describedabove in an acid-sensitivity assay, except that 0.1% peptonewater (pH 7.0) was supplemented with NaCl (1–10%). Thecomplemented and wild-type strains exhibited similargrowth, while the mutant showed inhibited growth(Fig. 1d, selected result). Therefore, the results showedthat expression of the  C. jejuni htrB   gene contributed to theosmotic resistance of the  S. typhimurium htrB   mutant. Role of the  C. jejuni htrB  gene in bile sensitivity of  S. typhimurium To examine whether expression of the  C. jejuni htrB   geneaffected bile sensitivity, the wild-type, mutant and com-plemented  Salmonella   strains were grown on LB agar platessupplemented with 2, 4, 6, 8 and 10% DOC. The results 257 htrB   and  C. jejuni   response to harsh environments  Downloaded from byIP: Thu, 22 Dec 2016 23:20:52 showed that the  C. jejuni htrB   gene could not complementthe bile sensitivity of the  S. typhimurium htrB   mutant, as themutant and complemented strains were able to grow in allDOC concentrations tested, while the wild-type strain wasable to grow in DOC up to 8%. Similarly, a bile-resistantphenotype of the  S. typhimurium htrB   mutant has also beenobserved by Sunshine  et al.  (1997). However, the study of bile sensitivity in other bacteria has shown inconsistentresults. The  H. influenzae htrB   mutant is more bile sensitivethan the wild-type strain (Lee  et al. , 1995), while the  E. coli htrB   mutant is more bile resistant than the wild-type strain(Karow & Georgopoulos, 1992; Sunshine  et al. , 1997). Sinceexpression of the  C. jejuni htrB   gene did not affect the bilesensitivity of the  S. typhimurium htrB   mutant, the  C. jejuni htrB  genemight nothavearolein bilesensitivity in C. jejuni  . Expression of the  htrB  gene in  C. jejuni   under stress environments To examine whether stress environments, including heat,acid,osmotic, oxidativeandbile stresses, affected expressionof the  htrB   gene in  C. jejuni  , quantitative real-time RT-PCR was performed. Before the quantitative data were accepted,we required three criteria. First, no PCR product should be Fig. 1.  Complementation experiments. WT, wild-type  S. typhimurium  strain SL1344; M,  S. typhimurium htrB  mutant; C,  S.typhimurium htrB  mutant carrying the functional  htrB  gene from  C. jejuni   HB 93-13; Na, NaCl concentration (%). (a)Morphology at different temperatures; (b) ability of bacteria to grow at high temperatures; (c) ability of bacteria to grow in highacidity; (d) ability of bacteria to grow in high osmolality. 258  Microbiology   153 V. Phongsisay, V. N. Perera and B. N. Fry
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