Characteristics of Helicobacter pylori natural transformation

Transcription

1 FEMS Microbiology Letters 188 (2000) 275^280 Characteristics of Helicobacter pylori natural transformation Dawn A. Israel *, Angela S. Lou, Martin J. Blaser Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine and VA Medical Center, A-3310 Medical Center North, Nashville, TN , USA Abstract Received 24 January 2000; received in revised form 9 March 2000; accepted 27 March 2000 For Helicobacter pylori, which exhibits substantial genetic diversity, many strains are naturally competent for transformation by exogenous DNA. To better understand the mechanism of natural transformation and its role in the generation of diversity, we sought to systematically identify factors important for natural transformation in H. pylori. We now show that the highest frequency of H. pylori transformation occurs when DNA is introduced prior to exponential phase growth, and that it is a saturable phenomenon. That transformation can be inhibited by DNA from Helicobacter (H. pylori and Helicobacter bilis) but not Escherichia coli suggests specificity based on DNA source. Finally, the cag island was determined to be unnecessary for high-frequency transformation. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Natural transformation; Genetics; Helicobacter pylori 1. Introduction Helicobacter pylori are Gram-negative bacteria that colonize the human stomach [1] and that have a role in peptic ulcer disease and gastric cancer [2]. Despite an overall conservation of most genes and their relationships with anking elements [3,4], H. pylori strains exhibit a high degree of genetic diversity [5^7]. Since markers associated with virulence have been identi ed [8^10], the genetic diversity of H. pylori strains is of clinical interest and techniques such as RFLP and RAPD-PCR utilize the heterogeneity to address questions about transmission and intrafamilial clustering of strains [6,11]. The generation of genetic diversity can be accelerated by the horizontal transfer of DNA. H. pylori strains show a panmictic population structure [12], indicating that horizontal DNA transfer is an important phenomenon [13]. Further, natural transformation is an important tool for the study of the genetics of H. pylori. Many strains of H. pylori are known to be naturally competent for transformation in vitro [14^16], but the mechanisms by which this occurs are not well understood. In this study, we sought to better characterize factors important for the natural transformation of H. pylori. * Corresponding author. Tel.: +1 (615) ; Fax: +1 (615) ; dawn.a.israel@vanderbilt.edu 2. Materials and methods 2.1. Bacterial strains and culture methods H. pylori strains used in this study are described in Table 1. Streptomycin-resistant mutants and ureb: :Kan r insertion mutants were made as described [17]. The mutation responsible for streptomycin-resistance was identi ed as A128G resulting in a lysine to arginine change at codon 43 in rps12 (data not shown). H. pylori was routinely grown on either TSA supplemented with 5% sheep blood (BBL) or Brucella agar (BBL) plates supplemented with 10% newborn calf serum (Intergen). For liquid culture, Brucella broth with 10% newborn calf serum and 20 Wg ml 31 vancomycin was used. Antibiotics were added to Brucella agar plates as necessary at the following concentrations: streptomycin, 20 Wg ml 31 ; spectinomycin, 20 Wg ml 31 ; or kanamycin, 25 Wg ml 31. Strains were grown at 37³C in 5% CO 2. Escherichia coli was grown on LB medium at 37³C [18] Natural transformation of H. pylori The transformation of H. pylori on plates was essentially as described [19]. Brie y, after growth on TSA plates for 2 days, cells were harvested into phosphate-bu ered saline (PBS), pelleted and resuspended in PBS. DNA was added to the cells which were then spotted on a / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (00)

2 276 D.A. Israel et al. / FEMS Microbiology Letters 188 (2000) 275^280 Table 1 Bacterial strains used in this study Strain designation Relevant characteristics a Other genotypes Origin (reference) caga icea vaca H. pylori HPK5 wild-type + 1 (s1a m1) Japan [20] HPK5 Strep r Kan r Strep r, php1 Kan r + 1 (s1a m1) this study HPK1 wild-type + 1 (s1a m1) Japan [20] HPK1 Strep r Kan r Strep r ureb: :Kan r + 1 (s1a m1) this study 84^183 wild-type + 2 (s1b m1) USA [29] 84^183 Strep r Kan r Strep r ureb: :Kan r + 2 (s1b m1) this study CH4 wild-type + 1 (s1a m2) China (this study) CH4 Strep r Strep r + 1 (s1a m2) this study 549/91 wild-type + 1 (s1a m1) Finland (this study) wild-type + 1 (s1a m1) UK [4] vcag cag PAI deletion, Cam r 3 1 (s1a m1) [31] HPK5 vcag cag PAI deletion, Cam r 3 1 (s1a m1) this study E. coli C600 F 3 thr-1 leub6 thi-1 lacy1 supe44 rfbd1 fhua21 [32] H. bilis ATCC wild-type [30] a Strep r, streptomycin-resistant; Kan r, kanamycin-resistant; Cam r, chloramphenicol-resistant. non-selective plate. After incubation overnight at 37³C, 5% CO 2, the cells were harvested into PBS and appropriate dilutions were inoculated onto selective and non-selective plates. After incubation for 4 days, colonies were counted and transformation frequencies calculated. Unless indicated otherwise in Section 3, 15 Wl of puri ed (2 ng Wl 31 ) or crude donor DNA was added to the cells. For saturation experiments, the amount of DNA added varied from 0 to 1000 ng (0, 3, 10, 30, 100, 300 and 1000 ng) as indicated in Fig. 1. In the competition experiments, 20 ng DNA from HPK5 Strep r Kan r was mixed with 0^2000 ng of DNA from either HPK5, Helicobacter bilis (ATCC 51631) or E. coli (C600) prior to the addition of DNA to cells. For transformation in liquid medium, strain HPK5 from two 48-h plates was harvested into 1 ml PBS, pelleted and resuspended in 1 ml Brucella broth. Brucella broth (25 ml) in a 250-ml ba ed ask was inoculated with the cell suspension and incubated at 37³C, 5% CO 2, with shaking. After 48 h, 2 ml of culture was used to inoculate 50 ml fresh Brucella broth in 500-ml ba ed asks. At 0, 8 or 23 h of growth, 10 Wg chromosomal DNA from HPK5 Strep r Kan r was added and the culture was grown with shaking at 37³C, 5% CO 2. At various times during incubation, 1-ml aliquots were removed, the optical density at 600 nm (OD 600 ) was measured and samples were assayed for streptomycin-resistant transformants and viable count as described above Preparation of chromosomal DNA Table 2 Frequency of transformation of H. pylori strains with homologous and heterologous chromosomal DNA a Recipient H. pylori strain Source of DNA HPK5 Strep R Kan R HPK1 Strep R Kan R CH4 Strep R Chromosomal DNA was prepared for use in transformation experiments by two methods. The rst method included CTAB as described [20] to extract puri ed DNA from the bacterial strains. The second method was used to generate a crude source of DNA. Cells were harvested into 1 ml PBS from plates after 2 days of growth and pelleted by centrifugation at 8500Ug for 5 min. The 84^ /91 Strep R Kan R Strep R HPK5 Strep R Kan R (puri ed) HPK5 119 þ 46 b 44 þ þ þ þ þ 2970 HPK1 12 þ 6 17 þ 4 12 þ 6 13 þ þ þ 52 CH4 7 þ 4 26 þ þ þ þ þ 86 84^ þ þ þ þ þ þ þ 6 24 þ 16 7 þ 1 12 þ 5 29 þ þ 326 a Transformation frequency is reported as mean transformantsu10 36 Wg 31 cfu 31 þ S.E.M. b Transformation with DNA from the heterologous (but antibiotic-resistant) strain underlined.

3 D.A. Israel et al. / FEMS Microbiology Letters 188 (2000) 275^ cell pellet was discarded, 500 Wl of the supernatant was transferred to a new tube, 50 Wl of chloroform was added, the solution was mixed by brie y shaking and the tube was incubated on ice for 5 min. After centrifugation at 8500Ug for 5 min, the aqueous phase was removed and used as a source of DNA. Both puri ed and crude DNA were quantitated spectrophotometrically. 3. Results 3.1. Transformation of H. pylori by chromosomal DNA from homologous and heterologous strains We rst sought to determine whether the H. pylori strain from which source DNA was isolated would a ect transformation frequencies. For these experiments, three strains from Asian patients and two strains from Western patients were used as recipients, and the donor DNA was isolated from streptomycin-resistant H. pylori strains derived from the wild-type strains. We examined transformation in which the DNA and cells were from related H. pylori strains (homologous) or were unrelated (heterologous). Each of the strains tested was naturally competent and could be transformed by crude preparations of DNA from both related and unrelated strains of H. pylori (Table 2) and there was little di erence in transformation frequency for each strain tested based on whether the DNA was homologous or heterologous. Similarly, there was little di erence in the frequency with which each DNA preparation transformed the recipient strains. These data indicated that H. pylori has no strong barriers to transformation by chromosomal DNA with point mutations. Since in the above transformation experiments, we used crude preparations of DNA, we next sought to determine whether the use of puri ed DNA would improve transformation e ciency, or might reveal di erences in transformation frequencies when homologous or heterologous DNA was used. Comparing crude and puri ed DNA from strain HPK5 Strep r Kan r to transform recipient H. pylori strains to streptomycin-resistance, the use of the puri ed DNA was more e cient than the crude DNA (Table 2). Although the homologous transformation was the most e cient when puri ed DNA was used, the recipient strains generally di ered less than one log 10 in their transformation frequencies Saturation of transformation frequency To determine whether the maximum transformation frequency was limited by the available DNA concentration, increasing amounts of DNA were used to transform a constant number of cells of strain HPK5. As little as 0.6 ng of chromosomal DNA per 10 8 cells was su cient to produce a high number of transformants (Fig. 1A) and Fig. 1. H. pylori transformation by exogenous DNA is saturable and can be inhibited with unmarked Helicobacter DNA. (A) Increasing amounts of DNA (0^1000 ng) from H. pylori HPK5 Strep r Kan r were used to transform wild-type strain HPK5 (mean cfu, 8.7U10 6 þ 4.1U10 6 ) to streptomycin resistance. The data shown are from three independent experiments. (B) A constant amount of puri ed DNA (20 ng) from H. pylori HPK5 Strep r Kan r was mixed with increasing amounts of puri ed DNA from wild-type strain HPK5, H. bilis strain or E. coli strain C600, and was used to transform wild-type strain HPK5 (mean cfu, 3.2U10 7 þ 3.78U10 6 ) to streptomycin resistance. Symbols indicate source of competitor DNA as follows: squares, H. pylori strain HPK5; diamonds, H. bilis strain 51631; circles, E. coli strain C600. The data shown are the mean of ve independent experiments. saturation occurred when approximately 6 ng of DNA was added to 10 8 cells. This is similar to the amount of DNA found to reach the maximum DNA uptake in Haemophilus in uenzae (5^10 ng per 10 8 cells) [21,22]. The maximum frequency of transformation for the strain of H. pylori tested was approximately 1.3U10 34 transformants (Wg DNA) 31 colony forming units (cfu) 31.

4 278 D.A. Israel et al. / FEMS Microbiology Letters 188 (2000) 275^280 Fig. 2. H. pylori transformation during growth in liquid medium. Wildtype H. pylori strain HPK5 was inoculated from a broth starter culture into liquid medium, then DNA from homologous strain HPK5 Strep r Kan r added, and growth of the culture continued. The results of three independent experiments (indicated by squares, diamonds and circles) are shown. A shows OD 600 (open symbols; 1 OD 600 = 9.5U10 7 þ 1.4U10 7 cfu ml 31 ) for each experiment as a function of time, where 0 h is the time of entry into exponential growth. B (closed symbols) indicates the number of transformants cfu 31 recovered for samples obtained at the indicated times Competition by DNA from wild-type Helicobacter and E. coli The speci city of H. pylori cells to be transformed by H. pylori DNA was assessed in competition experiments. Cells of strain HPK5 were incubated with a constant amount (20 ng) of DNA from HPK5 Strep r Kan r and increasing amounts of puri ed DNA from the homologous wild-type H. pylori strain (HPK5), H. bilis strain or E. coli strain C600 and transformation frequencies were determined (Fig. 1B). As expected, the addition of unmarked DNA from H. pylori resulted in fewer Strep r transformants, indicating competition. The addition of H. bilis DNA reduced the frequency of Strep r transformants to an extent similar to that for the H. pylori DNA, whereas the addition of E. coli DNA had little e ect. These results are consistent with the saturability of transformation and indicate that H. pylori has a mechanism for di erentiating Helicobacter DNA from the DNA of other organisms Transformation during growth in liquid culture To investigate the phenomenon of transformation in relation to H. pylori growth characteristics, we measured transformation frequencies of cells during growth in Brucella broth. The culture medium was inoculated with strain HPK5, puri ed DNA from strain HPK5 Strep r Fig. 3. Variation in transformation frequency by timing of DNA introduction into the culture. Wild-type strain HPK5 was inoculated from a broth starter culture into three asks of liquid medium and grown with shaking. At 0, 8 and 23 h after inoculation, puri ed DNA from strain HPK5 Strep r Kan r was introduced to the culture (indicated by arrows in A). Growth as measured by OD 600 (A) and number of streptomycinresistant transformants (B) is indicated over the course of time. Results shown are from a representative experiment; three independent experiments had similar results.

5 D.A. Israel et al. / FEMS Microbiology Letters 188 (2000) 275^ Kan r added, and aliquots removed periodically for determining OD and enumerating Strep r transformants cfu 31. The maximum recovery of transformants (viable cell) 31 occurred during mid- to late-lag phase (0^10 h prior to the onset of exponential phase growth) (Fig. 2). In other experiments, the same donor DNA was added 0, 8 or 23 h after cells of strain HPK5 were inoculated into fresh medium. The highest numbers of transformants cfu 31 were obtained when DNA was added at the time the culture was inoculated (t = 0 h). However, the peak did not occur until 24 h later, just before the onset of exponential growth (Fig. 3). Although DNA added 8 or 23 h after inoculation gave rise to transformants, the numbers were approximately 7- and 11-fold lower, respectively, than when DNA was added at t = 0. Whether the DNA was added at either t =0 or t = 8 h, the peak in the number of transformants cfu 31 occurred at approximately the same time (24 h), representing late-lag phase just prior to exponential phase. When the H. pylori DNA was introduced 23 h after inoculation of the recipient cell culture, the peak of transformation also occurred about 24 h later. These results indicate transformation is most e cient when DNA is present early after cells are subcultured to fresh medium and that the number of resulting transformants cfu 31 peaks prior to exponential phase E ect of cag island deletion on transformation frequency Since the cag island contains open reading frames that could encode products related to horizontal gene transfer (for example, HP0525, a trag homolog and HP0527, a virb10 homolog; [3,4]) as well as others of unknown function, we examined the competence of two strains in which the cag island had been deleted (Table 3). That the frequency of transformation is approximately the same in the wild-type strains and in the isogenic mutants indicates that none of the genes within the cag island is required for transformation, nor do they clearly facilitate the process. 4. Discussion In this study, we found that all of the H. pylori strains Table 3 Transformation of H. pylori wild-type and cag island deletion mutants Donor strain a Strep r transformants (Wg DNA) 31 cfu 31b Wild-type recipient vcag recipient Strep r 1.5U10 35 þ 4.9U U10 35 þ 1.1U10 35 HPK5 Strep r 4.0U10 33 þ 2.5U U10 33 þ 4.8U10 34 a Donor strains are spontaneous Strep r derivatives of wild-type and HPK5; 30 ng of DNA was used to transform homologous recipient strains. b Recipients are homologous to the donor strains, but are Strep s and are either wild-type or have had the cag island deleted. tested were transformed at high frequency by chromosomal DNA from other H. pylori strains, regardless of whether the source of DNA was homologous or heterologous. In contrast to a previous investigation [16], transformation of the strain studied was saturable and less than 10 ng per 10 8 cfu was su cient to yield the maximum frequency of transformants (Wg DNA) 31 cfu 31. It is possible that the amount of DNA required for saturation is strain-speci c, that saturation occurs only when homologous DNA is used, or that conducting the experiments on solid medium (this study) as opposed to broth [16] might a ect results. The maximum transformation frequency determined suggests that, even when DNA is in excess, only a fraction of the H. pylori cells in a culture are able to be transformed. That transformation of strain HPK5 was inhibited by the addition of unmarked DNA from H. pylori or H. bilis, but not from E. coli, suggests a rate-limiting step in H. pylori transformation that is speci c for Helicobacter DNA. These results are similar to those that led to the identi cation of uptake sequences required for e cient transformation of H. in uenzae [21,23]. Whether such uptake sequences are required for H. pylori transformation has yet to be determined. As has been demonstrated for other naturally competent bacteria, including H. in uenzae, the frequency of transformation is related to the growth phase [24]. That the frequency of transformation was greatest when DNA was added simultaneously with the bacterial subculture suggests that the cells are most receptive to exogenous DNA when they are emerging from stationary phase. The second, smaller peak of transformants cfu 31 may be related to the lysis of cells, releasing their DNA, which then would be available to transform other cells. From our experiments with crude preparations of DNA, we know that extracellular DNA exists, at least in older plate cultures of H. pylori (Table 2). Although the cag island contains many genes which by homology could be related to horizontal DNA exchange [3,4], that they are not required for H. pylori transformation indicates that other loci are critical. Two unlinked loci required for H. pylori transformation have been identi ed thus far (dpra and comb) [19,25,26]. The high degree of H. pylori genetic diversity observed [5^7,27,28] and the natural competence of H. pylori suggest that transformation may be an important H. pylori mechanism for horizontal DNA exchange under natural conditions. Studies to better characterize the mechanisms of natural H. pylori transformation should ultimately lead to an improved understanding of how chromosomal recombination may contribute to both the genetic diversity observed and the tness of the recipient cells. Acknowledgements This work was supported in part by R01 DK53707 from

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