94 M. Braëutigam et al. / FEMS Microbiology Letters 155 (1997) 93^98 subjected to a spore-inactivating process, e.g. autoclaving, ultra-high temperatu

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1 FEMS Microbiology Letters 155 (1997) 93^98 Evidence for natural transformation of Bacillus subtilis in foodstu s Martin Braëutigam, Christian Hertel *, Walter P. Hammes Institut fuër Lebensmitteltechnologie, Universitaët Hohenheim, D Stuttgart, Germany Received 14 July 1997; revised 7 August 1997; accepted 11 August 1997 Abstract The effect of foodstuffs on the natural transformation of Bacillus subtilis was investigated. As examples of complex food matrices milk with various fat contents as well as chocolate milk were used. The frequencies of transformation varied with the fat content and ranged between 3.8U10 34 and 1.4U Highest frequencies of about 3U10 33 were observed in chocolate milk with 1.5% fat. Development of competence was observed in chocolate milk, resulting in maximal transformation frequencies upon incubation for 10^12 h at 37³C. Keywords: Natural transformation; Development of competence; Bacillus subtilis; Foodstu ; Chocolate milk 1. Introduction * Corresponding author. Tel.: +49 (711) ; Fax: +49 (711) ; hertel@uni-hohenheim.de Bacterial gene transfer by natural transformation is a physiologically and genetically determined property of a number of bacterial strains [1]. In Bacillus subtilis competence is expressed post-exponentially in de ned minimal media [2]. In complex media B. subtilis acquires transformability at extremely low levels only, and the frequency of transformation is strongly a ected by the nature of the exogenous DNA. For example, the most e cient substrate is a high-molecular-weight DNA with sequences homologous to the host genome. To a lower extent plasmid DNA is a further substrate but its utilisation becomes more e ective when it contains homologous sequences that permit integration into the host genome [2,3]. Natural transformation was shown to take place in habitats such as soil and water. Depending on the nature of these habitats, great variations occur with regard to the physiological activities of the bacteria and the bio-availability as well as the persistence of exogenous DNA and thus to the transformation frequencies [2,4]. Foods are man-made niches of great variability in their ecological characteristics, and in previous investigations it has been shown that foodstu s permit an e cient natural gene transfer to take place by conjugation and transduction events among the associated bacteria [5,6]. On the other hand, no data are available on natural transformation in foods. B. subtilis is a ubiquitous organism contaminating food raw materials, and the endospores of this organism can be found in virtually all foods that have not been / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S (97)

2 94 M. Braëutigam et al. / FEMS Microbiology Letters 155 (1997) 93^98 subjected to a spore-inactivating process, e.g. autoclaving, ultra-high temperature (UHT) treatment. Thus, B. subtilis is commonly found in pasteurised milk and dairy products [7]. Moreover, B. subtilis is also used for production of the fermented soybean food natto [8]. It was the aim of our investigation to study natural transformation in complex food matrices. As an example of a naturally competent organism B. subtilis was chosen and homologous chromosomal DNA was used as a substrate for transformation. 2. Materials and methods 2.1. Bacterial strains and culture conditions The strains used were B. subtilis 0G1 and its auxotrophic derivative B. subtilis 1G20 (trpc2) [9]. The organisms were grown in minimal salts medium [10] with supplements, and stock cultures were prepared as described by Bron and Venema [9]and van Sinderen and Venema [11]. Minimal agar was prepared according to Mulder and Venema [12] DNA isolation, preparation of competent cells, and transformation experiments The isolation of DNA from B. subtilis 0G1, the preparation of competent cells from B. subtilis 1G20 and their transformation were performed as described by Bron [13]. The transformation protocol Table 1 The e ect of media on the transformation frequencies of B. subtilis Transformation medium Transformation frequency a Mean S.D. UHT milk (0.3% fat) 1.2U U10 34 UHT milk (1.5% fat) 1.4U U10 34 UHT milk (3.5% fat) 3.8U U10 34 Pasteurised milk (1.5% fat) 1.4U U10 34 Chocolate milk (1.5% fat) 2.5U U10 34 Tap water (sterile ltered) 1.4U U10 35 Competence medium 3.5U U10 34 a Mean values are based on three independent transformation experiments. Fig. 1. E ect of de ned food ingredients on the frequency of transformation. In the standard assay B. subtilis was transformed with chromosomal DNA (5 Wg ml 31 ) in the precense of increasing concentrations of BSA (b) or casein (F). was modi ed as follows. Cells employed in the transformation assay were taken as a 100 Wl aliquot from a frozen stock. After centrifugation the cells were resuspended in minimal salts medium. Transformation experiments were performed in a total volume of 200 Wl. Chromosomal DNA was added to obtain a nal concentration of 5 Wg ml 31. The mixture (ph 7.0) was incubated at 37³C for 30 min. The uptake of DNA was terminated by the addition of 5 Wl ofa solution containing 2 mg ml 31 DNase I (Boehringer Mannheim) and further incubation for 5 min. The transformation frequencies determined in that standard assay served as the basis for comparison with the results obtained in experiments with foodstu s. The following foodstu s were employed: sterile ltered bovine serum albumin (BSA, Sigma) and casein hydrolysate (Difco Laboratories); commercial UHTtreated milk with fat contents of 0.3, 1.5 and 3.5% respectively; pasteurised milk (1.5% fat); and commercial chocolate milk (1.5% fat). Transformants were detected on minimal agar without tryptophan. The total counts were determined by surface plating on minimal agar with tryptophan. Transformation frequencies were calculated as the ratio of transformants to bacterial counts (cfu).

3 M. Braëutigam et al. / FEMS Microbiology Letters 155 (1997) 93^98 95 Fig. 2. E ect of chromosomal DNA on the transformation frequency of B. subtilis in UHT milk with 1.5% fat (b) and in the standard assay (E) Development of competence in growth medium, UHT milk and chocolate milk Competence studies were performed in growth medium, UHT milk and chocolate milk. In a rst set of experiments B. subtilis was grown overnight in minimal salts medium [11]. Aliquots of 2 ml were centrifuged and the cells were resuspended in 30 ml each of growth medium, UHT milk (1.5% fat) and chocolate milk (0.3 and 1.5% fat). The cultures were incubated in triple-ba ed Erlenmeyer asks at 37³C with agitation. For determining the status of competence, aliquots of 300 Wl were removed every hour and subjected to transformation experiments with 0.2 Wg of DNA. The uptake of DNA was terminated as described above. In a second series of experiments the cells were pre-grown in chocolate milk (1.5% fat), and a 1% aliquot of the culture was used to inoculate 30 ml of the same chocolate milk. Incubation and determination of the development of competence were performed as above. To determine the e ect of foodstu s on the transformation a standard assay with B. subtilis was developed and de ned food components were added to the incubation mixture. The addition of BSA was limited by its low solubility in comparison to casein. As shown in Fig. 1, the increase of the concentration of BSA from 0.5 to 5% reduced the transformation frequencies about 4-fold. On the other hand, concentrations of casein of up to 10% exerted a positive e ect on the transformation whereas higher concentrations reduced the frequency. The maximum frequency of 2.3U10 33 was obtained with 5% casein which value is about 6-fold above the control. Experiments (data not shown) with added glucose, sucrose, glycerol or starch revealed that the assay tolerated de ned concentrations of these components (e.g. 10% glucose or sucrose) but higher concentrations generally reduced the transformation frequencies Transformation of B. subtilis in milk As an example of a complex food matrix milk was chosen. Aliquots of 100 Wl from the frozen stock of competent cells were centrifuged, and the cells were resuspended in 1 ml UHT milk (1.5% fat). To determine the e ect of the DNA concentration on the transformation frequency, increasing DNA concentrations were added. As control the dependence of transformation on DNA concentration was also determined in the standard assay. As shown in Fig. 2, 3. Results 3.1. The e ect of foodstu s on the transformation of B. subtilis Fig. 3. Persistence of B. subtilis DNA in UHT milk (1.5% fat) at 8³C (b) and 20³C (F). DNA (0.5 Wg ml 31 ) was pre-incubated at the given temperatures up to 12 days before competent cells were added and the transformation frequencies determined.

4 96 M. Braëutigam et al. / FEMSMicrobiology Letters 155 (1997) 93^98 Fig. 4. Development of competence of B. subtilis in minimal salts medium (A), UHT milk (B), and chocolate milk (C and D). The media were inoculated either with cells grown in minimal salts medium (A^C) or in chocolate milk (D). Solid symbols: viable counts; open symbols: transformation frequencies. 6 the transformation frequencies obtained in milk and in the standard assay were closely similar. In milk the threshold value for unambiguous detection of transformation was one order of magnitude above the corresponding DNA concentration in the standard assay. To investigate the transformation of B. subtilis in milk experiments were performed with UHT milk, pasteurised milk and a chocolate milk. As compiled in Table 1, the transformation frequencies obtained in UHT milk (0.3 and 1.5% fat), pasteurised milk (1.5% fat), and chocolate milk (1.5% fat) had increased about 10-fold above the value of the control (performed in competence medium [11]). Highest frequencies of about 3U10 33 were obtained in the chocolate milk. In UHT milk with 3.5% fat the frequencies were close to the control. The persistence of chromosomal DNA in UHT milk (1.5% fat) at 8³C and 20³C was investigated by determining its potential to transform competent cells of B. subtilis. For this purpose, 0.5 Wg ml 31 DNA was added to the milk and pre-incubated at 8 or 20³C for up to 12 days before transformation experiments were carried out. As shown in Fig. 3, the pre-incubation at 20³C caused a strong decrease of the transformation frequency, and no transformants could be detected after 4 days. At 8³C the transformation frequencies remained nearly unchanged within the 5-day period, and decreased by one order of magnitude after 12 days Development of competence of B. subtilis in UHT milk and chocolate milk The development of competence of B. subtilis was investigated in UHT milk (1.5% fat), chocolate milk (0.3% and 1.5% fat) and, for purposes of control, in minimal salts medium. In series I of the experiments the media were inoculated with cells recovered from a pre-culture grown in minimal salts medium (Fig. 4A^C). In series II, the pre-culture was grown in

5 M. Braëutigam et al. / FEMS Microbiology Letters 155 (1997) 93^98 97 chocolate milk (1.5% fat) and an aliquot was taken as inoculum (Fig. 4D). From the time course of the development of competence (Fig. 4) it can be derived that B. subtilis cultured in chocolate milk achieved higher cell yields than in minimal salts medium or UHT milk (1.5% fat). From series I it is evident that in the control and in milk the competence is developed but is gradually lost upon further incubation (Fig. 4A,B). This loss is especially abrupt in milk. Remarkably, the transformation frequencies in chocolate milk acquired a second maximum after 11 h of incubation (Fig. 4C). With chocolate milk of 0.3% fat, virtually the same time courses were obtained (data not shown). Thus, the fat content does not appear to be a factor of importance for the development of competence. As shown in Fig. 4D, the use of the chocolate milk as an inoculum resulted in a time course that was closely similar to that depicted in Fig. 4C. The rst maximum of transformation frequencies is seen after 1 h of inoculation, and the values of the second maximum are about 10-fold below those of the rst. 4. Discussion Micro-organisms are common natural contaminants of foods. Their presence may be without any e ect or lead to spoilage or even poisoning of food. Finally, micro-organisms can exert useful e ects as fermentation ora. In all these cases the food matrix provides a microbial ecosystem of great variability and may permit very high physiological activity of the associated micro ora. This activity also includes e cient transformation as was revealed in our studies with B. subtilis. The speci c nature of the complex food a ected the transformation at the level of either the established competence that was developed under de ned conditions or the development of the competence itself. The multitude of food ingredients contribute in speci c ways to the transformation of competent cells (Fig. 1). The stimulatory e ect of casein hydrolysate on the transformation is consistent with the results of Young and Spizizen [14] and Wilson and Bott [15]. Virtually the same value was obtained in complex foods, such as milk (Table 1). It has been described that B. subtilis develops maximal competence in de ned minimal media whereas in complex media it is developed to an extremely low extent only [2]. As we could demonstrate, development takes place even in complex foods, such as milk and chocolate milk. However, the two foodstu s differ greatly in the maintenance of this property. Whereas in milk the competence of the cells is lost completely when the culture enters the stationary phase, in chocolate milk a second peak of competence occurs in that growth phase. We have not investigated which constituent(s) of the chocolate milk ingredients were crucial for this e ect. Foods may, furthermore, vary greatly in their potential to render external DNA unavailable for transformation. An example was seen in UHT milk. Our observation is consistent with the assumption that even in that strongly heat-treated substrate nucleases are present that degrade DNA upon incubation at ambient temperatures. Our ndings provide a basis to study the fate of DNA and its speci c forms or sequences in de ned foods providing optimum conditions for natural transformation. The results obtained in our studies are consistent with data available for natural gene transfer in food by conjugation and transduction, for which an e ect of the food matrix was shown [5,6]. This knowledge is considered to contribute to a better understanding of microbial interactions and the characterisation of the `man-made ecosystem' food. Acknowledgments We thank Simone Esna-Ashari for excellent technical assistance. We are also grateful to G. Venema and his group for providing the strains and the introduction in the transformation technique at Groningen University. This work was supported by Bundesministerium fuër Bildung, Wissenschaft, Forschung und Technologie Grant References [1] Lorenz, M.G. and Wackernagel, W. (1994) Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev. 58, 563^602.

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