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1 ,2 ,3 Department of Infectious Diseases1 , Laboratory for Electron Microscopy2 and Second Department of Anatomy3 , Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo, Japan
4 Department of Internal Medicine, Keio University School of Medicine Shinjuku-ku, Tokyo, Japan
5 Department of Basic Laboratory Sciences, School of Allied Health Sciences, Osaka University, Yamadaoka, Suita, Osaka, Japan
6 Department of Microbiology, Kunming Medical School, Kunming, Republic of China
Correspondence
Takako Osaki
osaki{at}kyorin-u.ac.jp
Received 4 April 2006
Accepted 18 July 2006
Abbreviations: AI-2, type 2 autoinducer; qRT-PCR, semi-quantitative real-time reverse transcription PCR.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Helicobacter pylori has been identified as the aetiological agent of chronic active gastritis, peptic ulcer disease (Blaser, 1992; Graham, 1989), gastric adenocarcinoma (Parsonnet et al., 1991) and mucosal-associated lymphoid tissue (MALT) lymphoma (Wotherspoon et al., 1993). H. pylori has flagellar motility and is able to move through the viscous mucus gel lining the epithelium of the human stomach, thus leading to chronic infection of the human gastric mucosa. H. pylori produces extracellular signalling molecules related to AI-2, and production of AI-2 is dependent on luxS function (Forsyth & Cover, 2000; Joyce et al., 2000). These authors have reported that the production of AI-2 by luxS is growth-phase dependent, with maximal production occurring in the mid-exponential phase of growth. Furthermore, in their studies, maximal accumulation of AI-2 in the culture occurred in stationary phase. It has also been reported that the expression of H. pylori flaA is growth-phase dependent (Thompson et al., 2003), and that flaA transcription increases with culture density (Loh et al., 2004) at stationary phase. Mutating the luxS gene eliminates the growth-phase dependence of flaA, and growth-phase dependence is restored when the luxS mutant is complemented with wild-type luxS. Accordingly, we expected that the mutation of luxS of H. pylori might affect motility in the stationary phase, but not in exponential phase.
Several experimental animal models, such as monkeys (Drazek et al., 1994; Fujioka et al., 1997; Schauder et al., 2001), gnotobiotic piglets (Eaton et al., 1997), C57/BL6 mice (Lee et al., 1997; Kim et al., 2003) and Mongolian gerbils, are helpful in understanding the pathogenesis of human H. pylori infection. The Mongolian gerbil model, in which H. pylori is able to colonize long-term, is particularly easy to handle, and is useful as a small-animal model for the severe inflammation and obvious ulceration caused by H. pylori (Hirayama et al., 1996; Matsumoto et al., 1997; Yokota et al., 1991). The aim of this study is to investigate the effects of a defined luxS mutation on the characteristics of H. pylori and to determine whether the absence of AI-2 production allows H. pylori to infect and/or maintain infection in the gerbil gastric mucosa.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Construction of H. pylori luxS mutant. Primers HP105F2 (5'-GCTATTGCCTTGCAACAAATCCCCGC-3') and HP105R2 (5'-ATACTTAGGCGGGCATAGCGATG-3') were used to PCR-amplify a 1752 bp fragment containing the H. pylori luxS gene allele (luxS : : cat) with genomic DNA from strain TN2 (luxS : : cat) as template. The primers and genomic DNA of TN2 were kindly provided by Drs Ogura and Berg (Washington University, St Louis, MO). The fragment was then introduced directly into H. pylori 1402 by natural transformation via allelic exchange, and chloramphenicol-resistant colonies were isolated. Gene disruption in the mutant (designated HPKY08) was confirmed by PCR using the primer pairs HP105F2 and HP105R2, and HP02F (5'-CATACGAAAAAAGCCGCCC-3') and HP02R (5'-CTTGTCTGTGGGGATTGAG-3'), which yielded a PCR product of increased size. The mutation was also confirmed by Southern blot analysis with a probe containing the luxS gene. The mutant HPKY08 was used in all subsequent experiments.
Complementation of luxS mutant.
The gene luxS along with its promoter region was amplified using primers 105F2 and 105R2. This fragment was then cloned into the SspI site adjacent to the cat gene in pGEM. The resultant plasmid, pTKY451, was then transformed into the luxS– strain by natural transformation and was selected on Brucella-serum agar containing 5 µg kanamycin ml–1, and strain HpKY21, with luxS inserted in the chromosome, was obtained. Mutation of luxS and restoration of luxS function in the mutant were confirmed by AI-2 assay and Southern blot analysis of genomic DNA digested with SspI, using probes for the luxS gene, the chloramphenicol acetyl transferase gene (cat) and the kanamycin-resistance gene (aph) (Fig. 1
).
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). V. harveyi was grown at 30 °C in autoinducer bioassay (AB) broth medium (Hardie et al., 2003). Luminescence bioassay was performed as described previously (Forsyth & Cover, 2000). H. pylori or V. harveyi BB152 was grown in Brucella-serum broth, and culture supernatants were collected at various time points. Cell-free conditioned medium (CM) was prepared by centrifuging H. pylori or V. harveyi BB152 cultures in Brucella-serum broth at 8000 g for 15 min, followed by filtration (pore size 0.2 µm). CM preparations were routinely stored at –30 °C until use. The 18 h culture of V. harveyi BB170 was diluted 1 : 5000 in fresh AB medium. The experimental CM preparation (15 µl) was added to the diluted V. harveyi culture (135 µl), which was then incubated at 30 °C with agitation (180 r.p.m.). Next, 100 µl of this culture was added to a 96-well plate at various time points, and total luminescence was quantified using a luminometer (Mithras LB940, Berthold Technologies). Viable cell counts of V. harveyi BB170 were determined by culture using LM agar, and relative light units were calculated.
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The velocity of each H. pylori strain in Brucella broth containing 0.5 % glucose was also assessed using video systems. H. pylori was irradiated by the slanted dark-field light of a phase-difference condenser (c-c ph condenser, Nikon) and by observing scattered rays; H. pylori was detected as a bright point, which was not distinct in liquid medium against a light field. The obtained dark-field images were photographed at 30 frames per second sequentially by a 3CCD camera (DC-330, Dage-MTI), and transferred to computer in AVI file protocol through a capture card (SIM-PCI, DITECT). Motility was analysed using motility analysis software (DIPP-MOTION2D, DITECT). The movement of H. pylori on every frame was tracked for 1 s, and each coordinate was determined. The velocity of each H. pylori cell was calculated by the same software from coordinates using a frequency distribution chart. In one sample, 60 H. pylori bodies were tracked, and the mean velocities of wild-type and mutant strains were estimated and compared.
Ultrastructural study. For scanning electron microscopy, cultures of H. pylori strains in Brucella-serum broth were collected and attached to poly-L-lysine-coated cover slips (IWAKI) by centrifugation (1500 g, 5 min). Cells on the cover slips were washed twice in PBS and fixed with 2 % glutaraldehyde. Specimens were examined using a scanning electron microscope (JEOL JSM-5600LV).
Acid tolerance. Brucella broth supplemented with 10 % fetal calf serum was adjusted to pH 3 or pH 1 with HCl, and precipitated proteins were removed by filtration [acid-precipitated (AP) Brucella broth]. Next, 1–3x106 c.f.u. H. pylori ml–1 was inoculated into AP Brucella broth, with or without 30 mM urea, at pH 1 or 3. After 1 or 3 h incubation at 37 °C, viable bacterial counts were determined.
Animal experiments. Mongolian gerbils (MGS/sea; age 8 weeks; male) purchased from Seac Yoshitomi were fasted for 2 days and inoculated per os with 1–2x109 c.f.u. H. pylori TK1402 or HPKY08 (luxS– mutant) in Hanks' solution (Sigma) on two consecutive days. At each time point, four to five gerbils per group were sacrificed, and one half of the stomach was fixed in Carnoa's solution for histological analysis and the mucus layer of the other half was scraped off with a spatula and collected into 1 ml Hanks' solution. Of this 1 ml, 0.1 ml was inoculated onto M-BHM medium (Nikken Bio Medical Laboratory) and cultured at 37 °C for 7 days under microaerobic conditions. After the incubation period, gold colonies that exhibited urease activity were counted. Another 0.1 ml was treated with RNA protect (Qiagen) and stored at –80 C until used for RNA preparation.
The infective rate was determined by semi-quantitative real-time reverse transcription PCR (qRT-PCR), which detects expression of 16S rRNA (Nakagawa et al., 2005). Total RNA was extracted from samples by RNeasy Mini kit (Qiagen), according to the manufacturer's instructions. Contaminating chromosomal DNA was digested with DNase I (DNA-free, Ambion) [1 U (µg RNA)–1] for 20 min at 37 °C. One microgram of DNase I-treated total RNA was used with avian myeloblastosis virus (AMV) reverse transcriptase and random primers in a commercial reaction mixture (20 µl; AMV Reverse Transcription System, Promega) for first-strand complementary DNA synthesis.
Quantitative analysis was performed using the SYBR Green method. The generation of quantitative data was based on the different PCR kinetics of samples with various levels of target-gene expression. cDNA was amplified using PCR primers for H. pylori 16S rRNA, 16S2-F (5'-CGCTAAGAGATCAGCCTATGTCC-3') and 16SB2-R (5'-CCGTGTCTCAGTTCCAGTGTGT-3'). Amplification of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene on cDNA derived from Mongolian gerbils using the primers G3PDH-F (5'-ACCACAGTCCATGCCATCAC-3') and G3PDH-R (5'-TCCACCACCCTGTTGCTGTA-3') (RT-PCR Primer Set, TOYOBO) was used as a control and for standardization of target gene transcriptional activity data. For each primer set, PCR was performed in triplicate. Quantitative data were calculated from a standard curve generated by amplifying serial dilutions of a known quantity of amplicon. For this approach, the specificity of the PCR product was confirmed by dissociation curve analysis (7500 quantification program, Applied Biosystems).
ELISA. Serum antibodies from gerbils infected with H. pylori were assessed by ELISA at 3 months after infection, as previously reported (Nakagawa et al., 2005). A 96-well plate was coated with sonicated H. pylori TK1402 antigen (3 µg per well). Each serum sample was diluted 300-fold with PBS and added to the plates. Bound immunogloblins were detected with protein G-conjugated horseradish peroxidase (Sigma) and were developed using 0.1 % o-phenylenediamine and 0.035 % H2O2 in developing buffer (0.1 M citric acid, 0.07 M sodium phosphate dibasic). After incubation at room temperature for 5 min, the reaction was stopped by adding 50 µl 2(NH2)SO4. A490 was measured using a microplate reader (model 550, Bio-Rad).
Cell culture and characterization of H. pylori. Adhesion activity of H. pylori strains was assessed by flow cytometry, as previously reported (Osaki et al., 1997). AGS cells, originating from a gastric adenocarcinoma cell, were cultured in RPMI 1640 medium containing 10 % fetal calf serum under 5 % CO2 at 37 °C. Cells (5x105) were cultured with PKH-2-labelled HPKY08 (luxS– mutant) or the wild-type strain for 1 h at room temperature. The fluorescence intensity of the cells, which reflects adhesion activity, was analysed by flow cytometry (FACSVantage, Becton Dickinson).
Statistical analysis. Statistically significant differences were examined by Student's or Welch's t test.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). At 24, 33 and 54 h after inoculation, a cell-free culture of HPKY08 (luxS– mutant) did not yield bioluminescence in the assay, indicating no significant production of AI-2. However, cell-free culture of luxS-complemented HPKY21 showed AI-2 activity. These results clearly indicate that the luxS gene is responsible for the production of AI-2 in H. pylori. These data were in agreement with the results obtained by Forsyth & Cover (2000) and Joyce et al. (2000). In other words, H. pylori luxS plays an essential role in the production of extracellular signalling molecules.
Growth of luxS mutant
The microaerobic growth of HPKY08 (luxS– mutant) in Brucella-serum broth medium was compared to that of TK1402. In the exponential phase (0–16 h), the generation times for TK1402 and HPKY08 were 3.1±0.3 h and 4.0±0.8 h, respectively (P=0.1789, n=3). From the late-exponential to the stationary phase (24–64 h), the mean bacterial number (107.65±100.82 c.f.u. ml–1) for HPKY08 was slightly lower than that for TK1402 (108.33±100.68 c.f.u. ml–1) (P=0.1035, n=3). These results indicate a slight difference in growth characteristics between TK1402 and HPKY08 during the late-exponential phase. The generation time and mean bacterial number for HPKY21 complemented with luxS were 3.3±0.2 h and 108.23±100.81 c.f.u. ml–1, respectively, which were not significantly different from those of the TK1402 strain (P=0.6617 and 0.7886, respectively). These results suggest that the growth delay in H. pylori may be due to the effects of luxS mutation.
Motility on semi-solid agar and flagella formation of the HPKY08 (luxS– mutant) strain
Three strains of H. pylori were cultured on Brucella-serum medium containing 0.3 % agar. After 5 days of culture, halo diameters were measured. The HPKY08 (luxS– mutant) was motile, but the halo diameter (8.0±1.4 mm; n=4) after 5 days of culture was significantly smaller than that of TK1402 (12.3±2.0 mm; n=8) (P<0.05) (Fig. 2). The mean halo diameter of HPKY21 complemented with luxS was 14.0±1.2 mm (n=4), which was not significantly different from that of TK1402. These results confirmed that the reduction in H. pylori motility may be due to the effects of luxS mutation.
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). No morphological difference was observed between the two strains harvested after 15, 24 and 48 h cultivation.
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Effects of glucose on H. pylori motility
To evaluate the influence of glucose on the motility of H. pylori, HPKY08 (luxS– mutant) was cultured on Brucella semi-solid medium containing various concentrations of glucose (Fig. 4). On the medium containing 0.13–2 % glucose, HPKY08 motility was significantly inhibited. On the other hand, the motility of both wild-type and luxS-complemented strains was enhanced on medium containing 0.13–1 % glucose. At each concentration from 0 to 2 % glucose, the motility of the mutant was significantly lower than that of the wild-type and luxS-complemented strains (P<0.05) (Fig. 4). These results indicate that the motility of the luxS mutant is more sensitive to glucose.
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We demonstrated that the presence of glucose (0.13–2 %) strongly inhibited the motility of the luxS– mutant strain, although its growth was not inhibited by glucose (0.13–1 %) (data not shown). Kim et al. (2003) have reported that V. vulnificus shows increased signalling activity when cultured in the presence of 0.5 % glucose without growth enhancement. It has been reported that AI-2 production by virulent E. coli and S. Typhimurium is affected by glucose and pH level (Surette & Bassler, 1999). Similarly, in the present study, AI-2 production by the H. pylori wild-type TK1402 strain was significantly enhanced in the presence of 0.5 % glucose (data not shown). We also checked directly the movement of H. pylori strains in the Brucella-broth medium containing glucose, and showed that the velocity of the H. pylori luxS mutant strain was significantly lower than that of the wild-type and luxS-complemented strains.
Acid tolerance of the HPKY08 strain
Both wild-type and HPKY08 strains were prepared from stationary-phase cultures. In AP Brucella broth lacking urea, the number of both wild-type and HPKY08 (luxS– mutant) H. pylori decreased rapidly at pH 1, and no cells were viable after 1 h (data not shown). In medium containing 30 mM urea at pH 1, both the HPKY08 and wild-type strains recovered to 104 c.f.u. ml–1 at 1 h after inoculation of approximately 106 c.f.u. H. pylori ml–1. In the presence of 30 mM urea, both strains were maintained at 104 c.f.u. ml–1 for 3 h of incubation at pH 3. Similar results were obtained when the strains were in exponential phase (data not shown). These results show that there are no differences in survival between the wild-type and luxS– mutant strains under acidic conditions in the presence of urea.
Adhesion of HPKY08 H. pylori strain to MKN45 cells
The adhesion activities of HPKY08 (luxS–) and wild-type H. pylori, which were obtained from 3-day cultures on BHI agar supplemented with 7 % horse blood, were assessed by flow cytometry. The mean fluorescence intensities of the wild-type strain and HPKY08 (luxS–) strains were 2622.3±1400.8 and 1934.7±932.6, respectively, at m.o.i.=600, and there was no significant difference in adhesion activity between the two strains in any of the experiments (m.o.i.=60 or 6000). These results indicate that mutation of the luxS gene does not affect the adherence activity of H. pylori to AGS cells.
Colonization of Mongolian gerbils with HPKY08
In the Mongolian gerbil experiment, the detectable frequency of HPKY08 (luxS–) by culture was lower than that of the wild-type strain. At 1, 4 and 12 weeks after infection with TK1402, the rates of H. pylori-positive gerbils to total gerbils were 4/4, 4/5 and 3/5, and mean colonization numbers were 102.61±101.56, 103.70±100.91 and 105.05±102.51 c.f.u. (g gastric mucus)–1, respectively, upon culture using M-BHM agar plates (Fig. 5). In this culture method, the detection limit of H. pylori was 102.3 c.f.u. (g gastric mucus)–1. However, at 1, 4 and 12 weeks after infection with HPKY08, no H. pylori was isolated from the gastric mucus.
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). At 1 week after infection with H. pylori, the mean relative number of the luxS mutant [101.16±100.88 (g mucus)–1] was significantly lower than that of TK1402 [102.94±101.18 (g mucus)–1] (P=0.046). At 12 weeks after infection, the mean relative number of the luxS mutant was 102.24±100.28 (g mucus)–1, which was significantly lower than that of the wild-type strain [10 3.86±100.43 (g mucus)–1] (P<0.01). These data indicate that the colonization rate of the luxS mutant was significantly lower than that of TK1402 in gerbil stomachs, and that the luxS gene plays an important role in H. pylori infectivity in gerbils. For comparison of survival rates between the HPKY08 and TK1402 strains in the gerbil stomachs, we assessed the bacterial number in stomachs at 1 h after inoculation. The mean colonization number of HPKY08 [103.0±101.3 c.f.u. (g mucus)–1] was not significantly different from that of TK1402 (103.3±100.8 c.f.u. (g mucus)–1]. This suggests that there are no differences in survival in gerbil stomachs between the HPKY08 and TK1402 strains at this early time point.
Serum antibody titres from gerbils infected with H. pylori were assessed by ELISA at 12 weeks after infection. The ELISA value of serum antibodies from gerbils infected with HPKY08 (A490 0.24±0.90) was significantly lower than that of gerbils infected with TK1402 (A490 0.992±0.474; P<0.042). However, the antibody titre of gerbils infected with HPKY08 was higher than that of non-infected gerbils (A490 0.042±0.043; P<0.01).
On histopathological examination, no pathological changes were observed in the stomachs of gerbils infected with HPKY08 (luxS–) at 1 and 3 months after inoculation (Fig. 6a). However, severe gastritis was observed in gerbil stomachs infected with TK1402 at 3 months after infection (Fig. 6b). This suggests that the infectivity of the mutant strain is insufficient for induction of pathogenic changes in the gerbil stomach.
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In addition, it has been reported that motile strains are able to colonize regardless of their vacuolating cytotoxin activities, thus indicating that vacuolating cytotoxin is not associated with epithelial damage in the gastric mucosa (Iwao et al., 1999). In this study, it was shown that the luxS mutant had various virulence factors, such as vacuolating cytotoxin activity and hummingbird phenotype, caused by CagA protein injected into epithelial cells through type IV secretion machinery (data not shown). On histopathological examination, severe damage was observed in the gastric mucosa of gerbils infected with TK1402, but no damage was detected in the gastric mucosa of gerbils infected with HPKY08. It is therefore possible that induction of gastric damage depends on bacterial numbers in the stomach, and that the colonization potential is not associated with expression of these virulence factors.
H. pylori possesses numerous adhesive molecules, which are important for colonization in animal models. Yamaoka et al. (2002) have reported that the switch status of several genes, such as oipA, hopZ, hopO and hopP, encoding outer-membrane proteins influences the colonization of H. pylori in the stomachs of C57/BL mice. In their study, when two or more of these genes were ‘off’, colonization rates were markedly reduced compared with conditions under which all genes were ‘on’. However, in this study, it was shown that the adhesion of HPKY08 cells to MKN45 cells was almost identical to that of the wild-type TK1402 strain. In addition, there was no significant difference in the bacterial number in gastric mucosa at 1 h after inoculation between TK1402- and HPKY08-infected Mongolian gerbils. These results indicate that the decreased infectivity of the HPKY08 luxS mutant strain was not due to decreased adhesion activity.
Urease activity in H. pylori is required for its acid resistance, and thus for survival under the low-pH conditions of the stomach. In the present study, the urease activity of HPKY08 was as strong as that of TK1402 (data not shown). Both the wild-type TK1402 and the mutant HPKY08 strains survived at pH 3 in the presence of 30 mM urea. Therefore, the lower infectivity of HPKY08 is unlikely to be due to lower urease activity. In fact, there were no differences in the survival of the two strains in the stomachs of gerbils immediately after inoculation.
As shown by the qRT-PCR results (Fig. 5b) at 12 weeks after inoculation with HPKY08 (luxS–), considerable amounts of mRNA for H. pylori 16S rRNA were detected in the mucus of gerbil stomach, but isolation of H. pylori from gastric mucus was negative. We performed an animal experiment using the luxS-complemented strain HPKY21 in the Mongolian gerbil model. In the qRT-PCR assay, the number of colonizing H. pylori luxS-complemented HPKY21 strain cells was more than that of the luxS– mutant strain. In addition, there was no significant difference in the number of colonizing H. pylori between TK1402- and HPKY21-infected Mongolian gerbils (data not shown). These results support a hypothesis that the luxS mutation is involved in the colonization and persistent infection of H. pylori in the gastric mucosa of the Mongolian gerbil.
Serum antibody titres in gerbils infected with HPKY08 were slightly higher than those in non-infected gerbils. These data imply that HPKY08 colonized the gastric mucus of the gerbils, although the number of colonizing H. pylori cells was below the detection limit, 2x102 c.f.u. (g mucus)–1, for the culture-based assay. In gerbil stomachs, numerous other types of bacteria form colonies, and some of these may possess the luxS/AI-2 system. It is possible that the AI-2 produced by these bacteria affected the luxS mutant strain, thus allowing the mutant to survive in the gerbil stomach.
The biosynthetic pathway of AI-2 has recently been clarified (Schauder et al., 2001). AI-2 is generated from S-adenosylmethionine (SAM) in three enzymic steps. SAM is an essential metabolite used in central metabolism. Following methyl transfer from SAM to its various substrates, S-adenosylhomocysteine (SAH) is formed. SAH is a potent inhibitor of SAM-dependent methyl transferase, and thus must be eliminated. However, the luxS mutant is unable to convert the second product, S-ribosylhomocysteine (SRH), which is the substrate of LuxS, to AI-2 and homocysteine (third product). The accumulation of SAH in the mutant may inhibit bacterial cell growth. In fact, the viable cell number of HPKY08 in the stationary phase was lower than that of TK1402.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | J MED MICROBIOL | MICROBIOLOGY | J GEN VIROL | ALL SGM JOURNALS |
, dashed line), HPKY21 (luxS complemented; 
, dashed line) and TK1402 (wild-type; 
, solid line) strains. The strains were cultured on Brucella-serum semi-solid agar for 4 days, and the diameter of the halo was measured. **The mean diameter of halo was significantly lower than that of TK1402 (P<0.01).
