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Modified electroporation protocol for Lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool
Journal of Microbiological Methods 60 (2005) 353 – 363 www.elsevier.com/locate/jmicmeth
Modified electroporation protocol for Lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool Charlene K. Masona,1, Martin A. Collinsa,b, Keith Thompsonb,* b
a Queen’s University of Belfast, Newforge Lane, Belfast, BT9 5PX, Northern Ireland, UK Department of Food Science (Food Microbiology), Department of Agriculture and Rural Development for Northern Ireland, Newforge Lane, Belfast, BT9 5PX, Northern Ireland, UK
Received 25 March 2004; received in revised form 13 October 2004; accepted 18 October 2004 Available online 18 November 2004
Abstract Isolates of Lactobacillus spp. from a collection of potentially probiotic strains isolated from the crops of broiler chickens were found to be non-electrotransformable using published techniques. One strain of Lactobacillus salivarius was shown to develop electrocompetence when an overnight culture was incubated in fresh medium. The effect was enhanced if glycine was incorporated into the fresh growth medium. When these modifications were applied to a number of other crop isolates of Lactobacillus spp., electrocompetence could be detected in approximately half the strains tested. Two temperature sensitive plasmid vectors that had been used for the genetic modification of other lactic acid bacteria were introduced into a crop strain of Lb. salivarius. Both showed temperature sensitivity at 42 8C and above but were relatively stable at 37 8C. The genetic tool harbouring an IS element allowed the delivery of the plasmid to multiple independent sites in the host chromosome. Harnessing such genetic tools will facilitate the future genetic analysis of the host bacterium. D 2004 Elsevier B.V. All rights reserved. Keywords: Electroporation; Lactobacillus; Chicken crop; pG+host5:ISSI
1. Introduction Many bacteria of the genus Lactobacillus isolated from a variety of sources have been shown to * Corresponding author. Tel.: +44 28 90255616; fax: +44 28 90255009. E-mail address: [email protected] (K. Thompson). 1 Present address: Department of Surgery, Queen’s University of Belfast, Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland. 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.10.013
be transformable by electroporation and therefore are amenable to manipulation for genetic analysis (Mills, 2001) and, ultimately, strain improvement (Renault, 2002; Ahmed, 2003). In particular, strains associated with food fermentations have GRAS (Generally Recognised As Safe) status in the USA and are attractive hosts for recombinant proteins. A variety of techniques has been described for the electrotransformation of Lactobacilli (reviewed by Aukrust et al., 1995). In general, the strategies adopted have shown some similarities.
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The cells are grown to exponential phase in a medium containing a cell-wall-weakening agent such as glycine. They are then washed and resuspended to form a dense cell suspension in a buffer containing an osmotic stabilizer. Following administration of the electrotransforming pulse, cells are allowed to incubate in growth medium containing an osmotic stabilizer and then plated onto selective agar. The precise growth conditions and electroporation parameters used will normally have to be optimised for a given strain. By carefully manipulating these conditions, it has been possible to successfully electrotransform such previously recalcitrant species as Lb. delbrueckii subsp. bulgaricus (Serror et al., 2002). The crops of poultry are hosts to a variety of species of Lactobacilli (Edelman et al., 2002; Guan et al., 2003) including Lb. crispatus, Lb. reuteri, Lb. gasseri, Lb. salivarius, Lb. fermentum and Lb. acidophilus. In experiments designed to screen and identify potentially probiotic strains of Lactobacillus spp. from the poultry crop (Mason, 2003; Mason and Collins, in preparation), a number of isolates of Lactobacilli was obtained. These were characterised as belonging predominantly to species Lb. salivarius and Lb. crispatus. Experiments to transform these bacteria employed the electroporation protocols described by Thompson and Collins (1996) and Walker et al. (1996). Initially no strains tested were found to become electrocompetent. This finding is similar to that of Beasley et al. (2004) who confirmed that strains of Lb. crispatus isolated from the poultry crop and ileum electrotransformed at a low frequency or not at all. In the present paper, we report a modified electrotransformation protocol in which a pulse of glycine, rather than growth in glycine, was a key factor for eliciting the electroporation of a range of poultry Lactobacilli. The technique could also be used to enable electrotransformation of type strains of other species but did not improve the transformation frequency. It was shown that Lactobacilli from poultry could support the replication of both rolling circle and theta replicating plasmids, and shuttle vectors based on Lactobacillus spp. replicons. For some strains, the electrotransformation frequency was relatively high and this enabled the performance of a genetic tool to be studied in a novel host.
2. Materials and methods 2.1. Bacterial strains, culture media and growth conditions Strains of Lactobacilli adhering to the crops of 28or 35-day-old broiler chickens (Mason, 2003, Mason and Collins, in preparation) were isolated (Table 1). In addition, a number of type strains of different species of Lactobacilli and strains from different culture collections which had been used for electroporation experiments in other laboratories were included (Table 2). Cultures were maintained anaerobically on MRS agar (Oxoid CM361, Unipath, Basingstoke, UK) at 37 or 42 8C with tri-weekly sub-culture or stored frozen at 70 8C. Liquid cultures were prepared in MRS broth (Oxoid CM359) in 12 ml volumes in test tubes and incubated without agitation at 37 or 42 8C. Escherichia coli strain JM101 was used to amplify and maintain plasmids. Strain JM101 was maintained aerobically on LB agar or in LB broth (Sambrook et al., 1989). For the selection of electrotransformants, chloramphenicol (5 Ag/ml for Lactobacilli and 25 Ag/ ml for E. coli) or erythromycin (5 Ag/ml for Lactobacilli and 50 Ag/ml for E. coli) was used. 2.2. Characterisation of strains A total of 19 strains selected to represent the different variants identified previously by random amplified polymporphic DNA (RAPD) analysis was selected (Mason, 2003, Mason and Collins, in preparation; Table 1). These were characterised by 16S rDNA sequence analysis using primers SD-Bact0011-a-S-17 and SG-Lab-0677-a-A-17 as described by Heilig et al. (2002) and tentative identifications obtained by FASTA searches of prokaryotic DNA sequences at http://www. hgmp.mrc.ac.uk and BLASTN searches at http://www.ebi.ac.uk. 2.3. Electroporation protocol Overnight cultures of the strains under investigation were diluted into fresh pre-warmed MRS broth containing glycine (up to 1.1M) and sucrose (up to 1M). The optical density of the culture (A 600) was measured when required. Preparation of the bacteria by washing was as described previously (Thompson
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Table 1 Identification and electroporation of Lactobacilli isolated from poultry crops Strain number
16S rDNA identification
3 7 131 A63 Sn5 A78 136 A8 A28 A75 Sn1 Sn20 L 1 A7 Sn31 108 130 F
Lactobacillus crispatus Lb. crispatus Lb. crispatus Lb. crispatus Lb. crispatus Lb. crispatus Lb. salivarius Lb. salivarius Lb. salivarius Lb. salivarius Lb. salivarius Lb. salivarius Lb. salivarius Lb. johnsonii Lb. johnsonii Lb. vaginalis Lb. reuteri Lb. reuteri Lb. thermotolerans
a b
Transformation frequency (per Ag pFX3 DNA) 99% (Acc. No. AY335495)a 99% (Acc. No. AJ421225) 99% (Acc. No. AY335495) 99.5% (Acc. No. AF257097) 98.5% (Acc. No. AF257097) 98% (Acc. No. AY339182) 99.3% (Acc. No. AF420311) 98.9% (Acc. No. AF420311) 99% (Acc. No. AF420311) 99% (Acc. No. AF420311) 99.5% (Acc. No. AF335475) 98.9% (Acc. No. AF335475) 100% (Acc. No. AY137589) 99% (Acc. No. AE017206) 99% (Acc. No. AE017206) 98% (Acc. No. AF243177) 99% (Acc. No. LF16SRR1) 98% (Acc. No. LR16SRR1) 98.8% (Acc. No. AF317702)
1.2102 Not detectedb 7102 Not detectedb Not detectedb 1102 3103 1.5103 2102 25 8.4104 10 Not detectedb 4103 4102 Not detectedb Not detectedb Not detectedb 10
Best alignment with 16S sequences from database. Less than 10 transformants per Ag pFX3 plasmid DNA.
Table 2 Electroporation of type strains of Lactobacilli using standard and modified protocolsa Species
Lactobacillus salivarius subsp. salivarius Lb. salivarius subsp. salivarius Lb. salivarius subsp. salicinius Lb. crispatus Lb. crispatus Lb. casei Lb. delbrueckii subsp. lactis Lb. Lb. Lb. Lb. Lb.
sakei curvatus curvatus acidophilus plantarum
Lb. delbrueckii subsp. bulgaricus Lb. helveticus a
Designation
Origin
Electroporation frequenciesb Standard protocol
Modified protocol
NCDO 8817
Turkey faeces
80
100
NCDO 929 NCDO 1555 NCIMB 702172 NCIMB 702240 ATCC 393 LK1 (Derivative of strain WS97) LTH 681 LTH 683 LTH 684 ATCC 4356 NCFB 1193
Not recorded Not recorded Infant faeces Human urine Dairy strain Not recorded
5103 1105 Not detectedc Not detected 4.5103 6106
3103 6103 Not detected 40 2104 5107
Raw sausage Raw sausage Raw sausage Human origin Ensiled vegetable matter Bulgarian yogurt Cheese starter culture
Not detected Not detected Not detected Not detected 2104
Not detected 10 Not detected Not detected 4105
Not detected Not detected
Not detected Not detected
NCDO 1489 CNRZ 32
For published electroporation data, see Chassy and Flickinger (1987, Lactobacillus casei); Zink et al. (1991, Lb. delbrueckii subsp. lactis); Schmidt et al. (1999, Lb. sakei); Klein et al. (1993, Lb. curvatus); Walker et al. (1996; Lb. acidophilus), Thompson and Collins (1996; Lb. plantarum); Bhowmik and Steele (1993; Lb. helveticus). b Transformants per Ag pFX3 plasmid DNA. c Less than 10 transformants per Ag pFX3 plasmid DNA.
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and Collins, 1996). Briefly, 12 ml volumes of cultured cells were resuspended in 1 ml of ice-cold ultrapure water and washed twice. Cells were then resuspended in 1 ml of 50 mM EDTA and held on ice for 5 min then washed once in ultrapure water and twice in 0.3M sucrose solution before being resuspended 100 Al of 0.3 M sucrose. The concentrated cell suspension (120) was electroporated using a Gene Pulser electroporator (Bio-Rad, Hemel Hempstead, UK) in cuvettes with a 0.2-cm electrode gap (Flowgen, Ashby de la Zouch, UK) with up to 5 Al plasmid DNA (at a concentration of 100 Ag/ml). Except where stated, electroporation parameters were 1.5 kV, 200 V parallel resistance, and 25 AF capacitance. For phenotypic expression, the cells were diluted immediately into 1.9 ml of MRS broth in a 2-ml vial pre-warmed to 37 8C. After 3-h incubation, serial dilutions were plated onto MRS agar containing the selective antibiotic (chloramphenicol or erythromycin, as appropriate). 2.4. Plasmids Plasmids used for electrotransformation experiments with Lactobacillus salivarius Sn1 are listed in Table 3. Experiments to determine optimum electroporation conditions for chicken crop isolates used plasmid pFX3. The functionality of erythromycin resistant genetic tools pG+host5 (Biswas et al., 1993) and pG+host 5:ISS1 (Maguin et al., 1996) was determined using Lb. salivarius strain Sn1 as host.
salivarius strain Sn1 (pG+host5:ISS1) grown in MRS broth containing erythromycin at 37 8C was diluted 1/100 into fresh medium without selection to allow exponential growth to resume (ca. 150 min). The culture was then shifted to the restrictive temperature (42 or 48 8C) for 150 min to reduce the plasmid copy number, then diluted and plated onto selective agar at the restrictive temperature (to detect transposition) and non-selective agar at the permissive temperature (to determine the viable count). Colonies from selective agar at the restrictive temperature were analysed for the presence of free plasmid and integrated pG+host5:ISS1. Total DNA was extracted following growth of the bacteria at 42 8C with selection and in the presence of glycine (0.13M) using Promega Wizard Genomic DNA (Promega, Southampton, UK) extraction kits according to the manufacturers’ instructions. Undigested DNA and DNA digested with HinDIII were run on 0.8% (w/v) agarose gels, blotted onto Hybond N+ membranes and probed with ISS1 PCR product, or linearised pG+host5 labelled using an Enhanced Chemiluminescence kit (ECL; Amersham, Little Chalfont, UK) according to the manufacturers’ instructions. General molecular biology procedures were as described by Sambrook et al. (1989).
3. Results 3.1. Identification of crop isolates
2.5. Generation of integrants using plasmid pG+ host5:ISS1 These experiments were essentially as described by Maguin et al. (1996). An overnight culture Lb.
The tentative identifications for strains of Lactobacilli used for electroporation experiments based on 16S rDNA sequence analysis are listed in Table 1.
Table 3 Plasmids used for the electroporation of Lb. salivarius Sn1 Plasmid
Origin
Selective antibiotic
Transformation frequencya
pFX3 pNZ123 pLP825 pIL253 pLP3537
pFX1 (Xu et al., 1991). Rolling Circle Replication. pWV01 (de Vos, 1987). Rolling Circle Replication. Lactobacillus plantarum plasmid p8014-2; (Posno et al., 1991). pAMg1 deletion derivative (Simon and Chopin, 1988). Theta replication. Lactobacillus pentosus plasmid p353-1; (Posno et al., 1991).
Chloramphenicol Chloramphenicol Chloramphenicol Erythromycin Erythromycin
2104 1107 2104 2104 6104
a
Transformants per Ag plasmid.
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3.2. Optimisation of the electroporation frequency using poultry isolates of Lactobacillus spp. as hosts During initial experiments, none of the 19 strains tested was electrotransfomable using the methods of Thompson and Collins (1996) or Walker et al. (1996). However, by adjusting the concentration of glycine in the growth medium over a range from 0.13 to 1.1M, a few transformants (b5) were obtained from two strains; Sn1 and A78. One of these strains (Lb. salivarius Sn1) was selected for further experiments. 3.3. A pulse of glycine rather than growth in glycine improves electroporation efficiency Measurements of optical density and determination of viable counts suggested that little or no growth of the bacteria ensued following dilution into fresh MRS broth containing 1.1M glycine (data not shown). Therefore, in order to increase the number of transformants, the optimum dilution factor from an overnight culture was determined over the range 1/20 to 1/2.5. Increasing the inoculum size enhanced the transformation frequency with a 1/6 dilution being the optimum. Shorter incubation times with the higher inoculum were then evaluated. The bacteria developed electrocompetence within 30 min following exposure to fresh medium containing glycine with the transformation frequency peaking at 60 to 120 min (Fig. 1). In the absence of glycine, electrocompetence still developed after 30-min incubation although there was a subsequent decline after 120-min incubation. This was not affected by the presence of sucrose (0.3M; data not shown). Using a 90-min pulse, the effect of increasing concentrations of glycine in the growth medium was measured. It appeared (Fig. 2) that more consistent transformation frequencies were obtained in the presence of glycine. A glycine concentration of 0.27M was selected for routine electroporation.
Fig. 1. Appearance of electrocompetence in Lb. salivarius strain Sn1 following incubation in fresh growth medium. An overnight culture of Lb. salivarius strain Sn1 was diluted 1+5 into fresh MRS containing 1.1 M glycine. Cells were harvested at intervals and electrocompetence determined. Data are taken from three experiments.
which the Lb. salivarius Sn1 cells were exposed. The absolute transformation frequency had not reached a maximum with the addition of 0.5 Ag pFX3 DNA, which was the highest quantity used. The electroporation efficiency determined in nine experiments using a range of DNA concentrations (0.1–500 ng/100 Al cell suspension) varied from 1.9103 to 1.6105 transformants per Ag pFX3 DNA (mean 1.7104). 3.5. Effect of host modification of transforming DNA Total plasmid DNA obtained from pFX3-containing transformants of Sn1 was used to electroporate strain Sn1 in order to determine whether the electroporation frequency could be enhanced by using plasmid DNA carrying the host strain modification. Total plasmid DNA was extracted from a pFX3 transformant of host strain Sn1 and the plasmid DNA was purified by isopycnic centrifugation. This DNA transformed strain Sn1 at a mean frequency of 1.6105 transformants per Ag which is a six-fold increase over the mean frequency obtained with unmodified pFX3 DNA.
3.4. Effect of increasing the concentration of transforming DNA
3.6. Transformation of crop isolates using modified protocol
There was a linear relationship between electroporation frequency and the DNA concentration to
Using the electroporation protocol adapted for crop isolates and, in separate experiments, pFX3
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one of the Lb. salivarius strains tested became electrocompetent. 3.7. Comparison of techniques for the electroporation of type cultures
Fig. 2. Effect of increasing glycine concentrations on electroporation efficiency of Lb. salivarius strain Sn1. An overnight culture was diluted 1+5 into fresh MRS broth containing increasing concentrations of glycine. Cells were harvested after a 90-min incubation and electrocompetence determined. Datapoints are taken from six experiments.
A number of type strains of different species of Lactobacilli (Table 2) were electroporated using two methods. First, the method of Thompson and Collins (1996) in which an arbitrary concentration of glycine (1%; 0.13M) was selected for growth. Second, the modifications described in the present investigation were used. When data from the two protocols were compared, the range of strains electrotransformable and the frequencies obtained were similar (Table 2). 3.8. Electroporation of Lb. salivarius Sn1 using a range of vectors
plasmid DNA or pFX3 plasmid DNA carrying potential Lb. salivarius strain Sn1 modification, the electrocompetence of previously non-transformable strains was determined (Table 1). A total of 11 out of the remaining 18 strains was electrotransformable using the adaptations to the technique and a maximum of 0.5 Ag of transforming DNA. All but
Electrotransformation frequencies obtained using additional plasmids are shown in Table 3. All four additional plasmids were able to transform the host strain. A relatively high transformation frequency was obtained using plasmid pNZ123. When poorly transformable Lb. curvatus strain LTH683 was electro-
Fig. 3. Temperature sensitivity of plasmids pG+host5 and pG+host5:ISS1 in host strain Lb. salivarius Sn1. The graphs show the retention of the erythromycin-resistance marker for (a) pG+host5 n and (b) pG+host5:ISS1 at 37 8C (open symbols) and 42 8C (closed symbols). Plate counts in the presence and absence of erythromycin were compared at intervals. Twenty-four-hour incubation represented approximately 24 generations at 37 8C and 40 generations at 42 8C. Data are means of three experiments.
.
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porated using plasmid pNZ123, transformants were obtained at a frequency of 1105/Ag using the modified protocol compared with a frequency of 6102/Ag using the standard protocol. 3.9. Transfer of genetic tools into strain Sn1 Plasmids pG+host5 and pG+host5:ISS1 were transformed into strain Sn1 and their presence confirmed by agarose gel electrophoresis and Southern hybridization. Differences in stability of the plasmids following growth at 37 and 42 8C were determined (Fig. 3). Both vectors were unstable when maintained at 42 8C compared with growth at 37 8C. Erythromycin-resistant colonies obtained following growth of strain Sn1 (pG+host5:ISS1) at 42 8C were purified and maintained at 42 8C in the presence of the selective antibiotic. Total DNA extracts from erythromycin-resistant colonies obtained at 428 were probed with ISS1. A total of 8 out of 14 colonies tested revealed the presence of plasmid DNA consistent with free pG+host5:ISS1 plasmid. The six remaining colonies yielded DNA which showed some evidence for the presence of free plasmid DNA but also an additional signal consistent with the presence of ISS1 on the chromosome (data not shown). The location of the ISS1 element from these putative integrants was determined by filter hybridization of ISS1 probe with HinDIII digested total DNA. DNA from one integrant gave weak signals with bands consistent with the ISS1 (or pG+host5) probes (data not shown). The data for DNA extracted from the five remaining colonies are presented in Fig. 4. These show intense signals predominantly with bands at 5.3 and 1.4 kb (vector and insert). In addition, the presence of two additional HinDIII fragments for each integrant is consistent with insertion of pG+host5:ISS1 at five independent sites. The experiment was repeated using selection at 48 8C which is the maximum growth temperature for Lb. salivarius Sn1. In this instance, putative integrants were obtained at a frequency of 0.02% and it was noted that these varied significantly in colony size. Of 10 isolates tested, none showed the presence of free plasmid. However, HinDIII digestion of their total DNA gave identical signals with the ISS1 probe. In addition to the 5.3- and 1.4-kb signals associated with
Fig. 4. Analysis of ISS1 integration in Lb. salivarius strain Sn1. Erythromycin resistant colonies of Lb. salivarius strain Sn1 (pG+host5:ISS1) following growth at 42 8C were recovered and purified. HinDIII digested total DNA was probed with ECL-labelled ISS1 DNA. The upper arrow indicates the position of linearised pG+host5 (5.3 kb). The lower arrow indicates the position of the fragment containing the ISS1 element (1.4 kb). The five lanes show the positions of the different patterns obtained.
the vector and insert, there were bands at 2.4 and 1.0 kb (data not shown).
4. Discussion 4.1. Electroporation of Lactobacilli from the chicken crop The modified electroporation protocol described in this report facilitated the electroporation of 12 out of 19 previously non- or poorly transformable strains of Lactobacilli isolated from the chicken crop. Six of seven strains of Lb. salivarius isolated from chicken crops were rendered electrocompetent or had their transformation frequencies improved following application of the modified protocol. By contrast, three type-cultures of Lb. salivarius were successfully electrotransformed by both the standard method and the modified protocol. One of these
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strains (NCDO1555) is the same as ATCC11742 which was successfully also electrotransformed by Heng et al. (1999). A total of three out of six strains identified as Lb. crispatus were transformed. One of two type cultures was electrotransformable but at a low frequency. In a previous report on the electrotransformation of Lactobacilli isolated from the chicken crop, Beasley et al. (2004) reported transformation of Lb. crispatus strains (two of five strains tested) at low frequencies (b20 per Ag) using a vector based on a cryptic Lb. crispatus plasmid. Frequencies obtained for some strains in the present investigation were an order of magnitude higher (100 to 700 per Ag). Of the remaining six strains, the isolates identified as Lb. johnsonii were only electrotransfomable using the modified protocol. This species has previously been described as being poorly transformable (Walker and Klaenhammer, 1994). The remaining strains studied belonged to species which have not been reported to become electrocompetent. Of the type of cultures which were amenable to electrotransformation, the technique described herein was in most cases at least as effective as growing the cells for extended periods in the presence of a cell-wall-weakening agent. Although we have not presented direct evidence for the presence of a modification of DNA passaged through Lb. salivarius Sn1, the observation that plasmid pFX3 extracted from Sn1 transformants was able to transform some of the target organisms more efficiently than unmodified pFX3 DNA would be consistent with this argument. Furthermore, the use of appropriately modified plasmid DNA may be a key factor for the successful transformation of strains with a functional restriction system. The protocol described in the present report contrasts with those described by, for example, Aukrust et al. (1995) who recommended mid-log cultures, Serror et al. (2002) who used early stationary phase cultures, and Zink et al. (1991) and Aymerich et al. (1993) who reported that some strains of Lb. delbrueckii subsp. lactis and Lb. sakei, respectively, gave highest electroporation frequencies on entering stationary phase. It was observed that all of the chicken crop isolates involved in the present investigation had a propensity to clump when incubated in MRS broth. When the bacteria were sub-cultured into
fresh medium, the period of incubation at which electroporation frequency was at its maximum, the clumps tended to disperse. A trivial explanation for this would be that more cells were available as targets for electrotransformation. Alternatively, cells entering exponential growth phase could be more susceptible to penetration by the DNA, and that the electrocompetence is maintained by the addition of glycine to the medium. The specific effect of glycine on these bacteria has not been identified although Hammes et al. (1973) suggested that glycine weakens the cell walls of Lactobacilli by reducing the number of cross links resulting in the disruption of the balance in peptidoglycan turnover and the misincorporation of glycine-containing precursors. Data from our laboratory and that of Beasley et al. (2004) suggest that bacteria from the chicken GI tract are recalcitrant with regard to electrotransformation. Some isolates can be rendered electrocompetent, however, by manipulating the growth conditions. Furthermore, the data suggest that empirical selection of a suitable plasmid for electrotransformation of a given strain will be an important consideration for the study of poorly transformable strains. A clear understanding of the nature of electrocompetence in poultry Lactobacilli will require further work. In addition, it appears that the requirement for optimisation of electroporation conditions is not only species and strain specific but may also have to be applied to bacteria isolated from any particular ecological niche. 4.2. Application of genetic tools to the genetic analysis of Lactobacilli from the chicken crop Plasmids exhibiting intrinsic temperature sensitivity when maintained in novel hosts have been used to deliver genetic markers to specific sites on the chromosome (Bhowmik and Steele, 1993; Serror et al., 2003). The intrinsic temperature sensitivity of certain plasmids has also permitted the development of novel vector systems for Lb. acidophilus and Lb. gasseri (Russell and Klaenhammer, 2001) and Lb. curvatus (Neu and Henrich, 2003). Plasmids pG+host5 and pG+host5:ISS1 are temperature sensitive derivatives of a Lactococcal plasmid pWV01-based replicon and exhibit marked instability in Lactococci (Biswas et al., 1993; Maguin
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et al., 1996) when the temperature is shifted from 28 to 37.5 8C. However, these genetic tools are generally not effective in Lactobacilli. For example, data from our laboratory (Steudel, 1996) showed that the vectors were stable in Lb. plantarum NCFB1193 following incubation at 28 or 37 8C and did not show instability until the growth temperature was raised to 41 8C when there was a 10-fold drop in the number of colony-forming units retaining the plasmid marker after a 20-h incubation. There was no evidence for random integration of the vector during these experiments. Gory et al. (2001) used a pG+host5-derivative to deliver a genetic material to a plasmid-cured strain of Lb. sakei but found that the plasmid was stable after 100 generations at 30 8C. It has been reported (personal communications in Serror et al., 2003) that the vector was not thermosensitive in either Lb. casei or Lb. plantarum. However, Russell and Klaenhammer (2001) noted that pGhost+5 precursor plasmid pGK12 was intrinsically temperature sensitive at 438 C in Lb. gasseri and Lb. johnsonii with only 0.01% of colony-forming units being retained after 30 generations at the elevated temperature. Pridmore (in Klaenhammer et al., 2002) reported that pG+host-based vectors were sufficiently temperature sensitive to be used efficiently for advanced genetic analysis in Lb. johnsonii. Because Lb. salivarius strain Sn1 appeared to have an optimum growth temperature of approximately 42 8C, and a maximum growth temperature of 48 8C, it was possible that the pG+host-based vectors could be functional in this host. This proved to be the case. The plasmid erythromycin resistance marker from pG+host5 and pG+host5:ISS1 was recovered in only 0.01% of colony forming units after a 6-h incubation (12 generations) at 42 8C. By contrast, at 37 8C the marker was still present in 50% and 10% of colony forming units after 30 generations for pG+host5 and pG+host5:ISS1, respectively. Although these data contrast with results obtained using Lactococci as hosts (Maguin et al., 1996) where the transposition frequencies of 10 6 and V10 2 for pG+host5 and pG+host5:ISS1, respectively, were reported, the thermal stability data are consistent with the findings of Serror et al. (2003) for transposition in Lb. delbrueckii subsp. bulgaricus at 44 8C when using related vector pG+host9. Although Serror et al. (2003) considered it unlikely that an ISS1-containing
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plasmid was actually transposing in Lb. delbrueckii subsp. bulgaricus, the same plasmid containing other lactic-acid-bacteria-derived insertion elements was able to transpose and into a range of chromosomal locations. Data presented here show that in Lb. salivarius plasmid, pG+host5:ISS1 can deliver the ISS1 element from the plasmid to a range of sites in the bacterial chromosome. It is probable that the integration has resulted in tandem duplication (Maguin et al., 1996). This is supported by the observation that in four of the five integrations (Fig. 4), the intensity of the bands for the pG+host 5 and ISS1 sequences using the relevant probes was greater than for the putative flanking HinDIII fragments. Single integrations of the genetic tool would be more desirable in order to prevent possible rearrangements and to facilitate sequencing of the flanking regions. In order to reduce the residual, free plasmid in the integrants, the selection temperature was raised to 488 C. Although this had the desired effect, the integrants tested appeared to be identical. This result was unexpected. It is possible that all colonies tested represent independent integrations into the same site but an alternative explanation is that the isolates were clonal and represent the most competitively growing integrants at the elevated temperature. Indeed the isolates selected for this experiment tended to be the larger colony-forming variants among the population. Further experimental work will therefore be necessary to optimise the conditions for selection of integrants. In conclusion, the modified electroporation protocol described and the temperature sensitivity of the genetic tool will permit gene expression analysis in this important group of organisms to be analysed and for knock-out mutants to be created for genes of interest.
Acknowledgements The authors wish to thank Mrs. Christine Nicholson and Mr. Gerard Cottrell for the expert technical assistance at different times during these experiments. One of us (CM) acknowledges the financial assistance from a DARDNI post-graduate studentship.
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References Ahmed, F.E., 2003. Genetically modified probiotics in foods. Trends Biotechnol. 21, 491 – 497. Aukrust, T.W., Brurberg, M.B., Nes, I.F., 1995. Transformation of Lactobacillus by electroporation. In: Nickoloff, J.A. (Ed.), Electroporation Protocols for Microorganisms, Methods in Molecular Biology. Humana Press, New Jersey, pp. 201 – 208. Aymerich, M.T., Hugas, M., Garriga, M., Vogel, R.F., Montfort, J.M., 1993. Electrotransformation of meat lactobacilli. Effect of several parameters on their efficiency of transformation. J. Appl. Bacteriol. 75, 320 – 325. Beasley, S.S., Takala, T.M., Reunanen, J., Apajalahti, J., Saris, P.E.J., 2004. Characterization and electrotransformation of Lactobacillus crispatus isolated from chicken crop and intestine. Poultry Sci. 83, 45 – 48. Bhowmik, T., Steele, J.L., 1993. Development of an electroporation procedure for gene disruption in Lactobacillus helveticus CNRZ32. J. Gen. Microbiol. 139, 1433 – 1439. Biswas, I., Gruss, A., Ehrlich, S.D., Maguin, E., 1993. Highefficiency gene inactivation and replacement system for Grampositive bacteria. J. Bacteriol. 175, 3628 – 3635. Chassy, B.M., Flickinger, J.L., 1987. Transformation of Lactobacillus casei by electroporation. FEMS Microbiol. Lett. 44, 173 – 177. de Vos, W.M., 1987. Gene cloning and expression in lactic streptococci. FEMS Microbiol. Rev. 46, 281 – 295. Edelman, S., Westerlund-Wikstrfm, B., Leskel7, S., Kettunen, H., Rautonen, N., Apajalahti, J., Korhonen, T.K., 2002. In vitro adhesion specificity of indigenous Lactobacilli within the avian intestinal tract. Appl. Environ. Microbiol. 68, 5155 – 5159. Gory, L., Montel, M.-C., Zagorec, M., 2001. Use of green fluorescent protein to monitor Lactobacillus sakei in fermented meat products. FEMS Microbiol. Lett. 194, 127 – 133. Guan, L.L., Hagen, K.E., Tannock, G.W., Korver, D.R., Fasenko, G.M., Allison, G.E., 2003. Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis. Appl. Environ. Microbiol. 69, 6750 – 6757. Hammes, W., Schleifer, K.H., Kandler, O., 1973. Mode of action of glycine on the biosynthesis of peptidoglycan. J. Bacteriol. 116, 1029 – 1053. Heilig, H.G.H.J., Zoetendal, E.G., Vaughan, E.E., Marteau, P., Akkermans, A.D.L., de Vos, W.M., 2002. Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA. Appl. Environ. Microbiol. 68, 114 – 123. Heng, N.C.K., Bateup, J.M., Loach, D.M., Wu, X., Jenkinson, H.F., Morrison, M., Tannock, G.W., 1999. Influence of different functional elements of plasmid pGT232 on maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro and in vivo. Appl. Environ. Microbiol. 65, 5378 – 5385. Klaenhammer, T., Altermann, E., Arigoni, F., Bolotin, A., Breidt, F., Broadbent, J., et al., 2002. Discovering lactic acid bacteria by genomics. Antonie van Leeuwenhoek 82, 29 – 58.
Klein, J.R., Ulrich, C., Plapp, R., 1993. Characterization and sequence analysis of a small cryptic plasmid from Lactobacillus curvatus LTH683 and its use for construction of new Lactobacillus cloning vectors. Plasmid 30, 14 – 29. Maguin, E., Prevost, H., Ehrlich, S.D., Gruss, S.D., 1996. Efficient insertional mutagenesis in lactococci and other Gram-positive bacteria. J. Bacteriol. 178, 931 – 935. Mason, C.K., 2003. Investigation of the probiotic potential of bacteria isolated from chicken crops with respect to cellular adherence in vitro. PhD thesis. Queens University, Belfast. Mills, D.A., 2001. Mutagenesis in the post genomics era: tools for generating insertional mutations in the lactic acid bacteria. Curr. Opin. Biotechnol. 12, 503 – 509. Neu, T., Henrich, B., 2003. New thermosensitive delivery vector and its use to enable nisin-controlled gene expression in Lactobacillus gasseri. Appl. Environ. Microbiol. 69, 1377 – 1382. Posno, M., Leer, R.J., van Luijk, N., van Giezen, M.J.F., Heuvelmans, P.T.H.M., Lokman, B.C., Pouwels, P.H., 1991. Incompatability of Lactobacillus vectors with replicons derived from small cryptic Lactobacillus plasmids and segregational instability of the introduced vectors. Appl. Environ. Microbiol. 57, 1822 – 1828. Renault, P., 2002. Genetically modified lactic acid bacteria: applications to food or health and risk assessment. Biochemie 84, 1073 – 1087. Russell, W.M., Klaenhammer, T.R., 2001. Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl. Environ. Microbiol. 67, 4361 – 4364. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Schmidt, G., Hertel, C., Hammes, W.P., 1999. Molecular characterisation of the dnaK operon of Lactobacillus sakei LTH681. Syst. Appl. Microbiol. 22, 321 – 328. Serror, P., Sasaki, T., Ehrlich, S.D., Maguin, E., 2002. Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids. Appl. Environ. Microbiol. 68, 46 – 52. Serror, P., Ilami, G., Chouayekh, H., Ehrlich, S.D., Maguin, E., 2003. Transposition in Lactobacillus delbrueckii subsp. bulgaricus: identification of two thermosensitive replicons and two functional insertion sequences. Microbiology 149, 1503 – 1511. Simon, D., Chopin, A., 1988. Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 70, 559 – 566. Steudel, U., 1996. Introduction of the temperature sensitive vector pG+host5 into Lactobacillus plantarum NCFB1193. Diplomarbeit, Technische Universit7t Mqnchen. Thompson, K., Collins, M.A., 1996. Improvement in electroporation efficiency for Lactobacillus plantarum by the inclusion of high concentrations of glycine in the growth medium. J. Microbiol. Methods 26, 73 – 79. Walker, D.C., Klaenhammer, T.R., 1994. Isolation of a novel IS3 group insertion element and construction of an integration vector for Lactobacillus spp. J. Bacteriol. 176, 5330 – 5340.
C.K. Mason et al. / Journal of Microbiological Methods 60 (2005) 353–363 Walker, D.C., Aoyama, K., Klaenhammer, T.R., 1996. Electrotransformation of Lactobacillus acidophilus Group A1. FEMS Microbiol. Lett. 138, 233 – 237. Xu, F., Pearce, L.E., Yu, P.-L., 1991. Construction of a family of Lactococcal vectors for gene cloning and translational fusion. FEMS Microbiol. Lett. 77, 55 – 59.
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Modified electroporation protocol for Lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool Charlene K. Masona,1, Martin A. Collinsa,b, Keith Thompsonb,* b
a Queen’s University of Belfast, Newforge Lane, Belfast, BT9 5PX, Northern Ireland, UK Department of Food Science (Food Microbiology), Department of Agriculture and Rural Development for Northern Ireland, Newforge Lane, Belfast, BT9 5PX, Northern Ireland, UK
Received 25 March 2004; received in revised form 13 October 2004; accepted 18 October 2004 Available online 18 November 2004
Abstract Isolates of Lactobacillus spp. from a collection of potentially probiotic strains isolated from the crops of broiler chickens were found to be non-electrotransformable using published techniques. One strain of Lactobacillus salivarius was shown to develop electrocompetence when an overnight culture was incubated in fresh medium. The effect was enhanced if glycine was incorporated into the fresh growth medium. When these modifications were applied to a number of other crop isolates of Lactobacillus spp., electrocompetence could be detected in approximately half the strains tested. Two temperature sensitive plasmid vectors that had been used for the genetic modification of other lactic acid bacteria were introduced into a crop strain of Lb. salivarius. Both showed temperature sensitivity at 42 8C and above but were relatively stable at 37 8C. The genetic tool harbouring an IS element allowed the delivery of the plasmid to multiple independent sites in the host chromosome. Harnessing such genetic tools will facilitate the future genetic analysis of the host bacterium. D 2004 Elsevier B.V. All rights reserved. Keywords: Electroporation; Lactobacillus; Chicken crop; pG+host5:ISSI
1. Introduction Many bacteria of the genus Lactobacillus isolated from a variety of sources have been shown to * Corresponding author. Tel.: +44 28 90255616; fax: +44 28 90255009. E-mail address: [email protected] (K. Thompson). 1 Present address: Department of Surgery, Queen’s University of Belfast, Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland. 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.10.013
be transformable by electroporation and therefore are amenable to manipulation for genetic analysis (Mills, 2001) and, ultimately, strain improvement (Renault, 2002; Ahmed, 2003). In particular, strains associated with food fermentations have GRAS (Generally Recognised As Safe) status in the USA and are attractive hosts for recombinant proteins. A variety of techniques has been described for the electrotransformation of Lactobacilli (reviewed by Aukrust et al., 1995). In general, the strategies adopted have shown some similarities.
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The cells are grown to exponential phase in a medium containing a cell-wall-weakening agent such as glycine. They are then washed and resuspended to form a dense cell suspension in a buffer containing an osmotic stabilizer. Following administration of the electrotransforming pulse, cells are allowed to incubate in growth medium containing an osmotic stabilizer and then plated onto selective agar. The precise growth conditions and electroporation parameters used will normally have to be optimised for a given strain. By carefully manipulating these conditions, it has been possible to successfully electrotransform such previously recalcitrant species as Lb. delbrueckii subsp. bulgaricus (Serror et al., 2002). The crops of poultry are hosts to a variety of species of Lactobacilli (Edelman et al., 2002; Guan et al., 2003) including Lb. crispatus, Lb. reuteri, Lb. gasseri, Lb. salivarius, Lb. fermentum and Lb. acidophilus. In experiments designed to screen and identify potentially probiotic strains of Lactobacillus spp. from the poultry crop (Mason, 2003; Mason and Collins, in preparation), a number of isolates of Lactobacilli was obtained. These were characterised as belonging predominantly to species Lb. salivarius and Lb. crispatus. Experiments to transform these bacteria employed the electroporation protocols described by Thompson and Collins (1996) and Walker et al. (1996). Initially no strains tested were found to become electrocompetent. This finding is similar to that of Beasley et al. (2004) who confirmed that strains of Lb. crispatus isolated from the poultry crop and ileum electrotransformed at a low frequency or not at all. In the present paper, we report a modified electrotransformation protocol in which a pulse of glycine, rather than growth in glycine, was a key factor for eliciting the electroporation of a range of poultry Lactobacilli. The technique could also be used to enable electrotransformation of type strains of other species but did not improve the transformation frequency. It was shown that Lactobacilli from poultry could support the replication of both rolling circle and theta replicating plasmids, and shuttle vectors based on Lactobacillus spp. replicons. For some strains, the electrotransformation frequency was relatively high and this enabled the performance of a genetic tool to be studied in a novel host.
2. Materials and methods 2.1. Bacterial strains, culture media and growth conditions Strains of Lactobacilli adhering to the crops of 28or 35-day-old broiler chickens (Mason, 2003, Mason and Collins, in preparation) were isolated (Table 1). In addition, a number of type strains of different species of Lactobacilli and strains from different culture collections which had been used for electroporation experiments in other laboratories were included (Table 2). Cultures were maintained anaerobically on MRS agar (Oxoid CM361, Unipath, Basingstoke, UK) at 37 or 42 8C with tri-weekly sub-culture or stored frozen at 70 8C. Liquid cultures were prepared in MRS broth (Oxoid CM359) in 12 ml volumes in test tubes and incubated without agitation at 37 or 42 8C. Escherichia coli strain JM101 was used to amplify and maintain plasmids. Strain JM101 was maintained aerobically on LB agar or in LB broth (Sambrook et al., 1989). For the selection of electrotransformants, chloramphenicol (5 Ag/ml for Lactobacilli and 25 Ag/ ml for E. coli) or erythromycin (5 Ag/ml for Lactobacilli and 50 Ag/ml for E. coli) was used. 2.2. Characterisation of strains A total of 19 strains selected to represent the different variants identified previously by random amplified polymporphic DNA (RAPD) analysis was selected (Mason, 2003, Mason and Collins, in preparation; Table 1). These were characterised by 16S rDNA sequence analysis using primers SD-Bact0011-a-S-17 and SG-Lab-0677-a-A-17 as described by Heilig et al. (2002) and tentative identifications obtained by FASTA searches of prokaryotic DNA sequences at http://www. hgmp.mrc.ac.uk and BLASTN searches at http://www.ebi.ac.uk. 2.3. Electroporation protocol Overnight cultures of the strains under investigation were diluted into fresh pre-warmed MRS broth containing glycine (up to 1.1M) and sucrose (up to 1M). The optical density of the culture (A 600) was measured when required. Preparation of the bacteria by washing was as described previously (Thompson
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Table 1 Identification and electroporation of Lactobacilli isolated from poultry crops Strain number
16S rDNA identification
3 7 131 A63 Sn5 A78 136 A8 A28 A75 Sn1 Sn20 L 1 A7 Sn31 108 130 F
Lactobacillus crispatus Lb. crispatus Lb. crispatus Lb. crispatus Lb. crispatus Lb. crispatus Lb. salivarius Lb. salivarius Lb. salivarius Lb. salivarius Lb. salivarius Lb. salivarius Lb. salivarius Lb. johnsonii Lb. johnsonii Lb. vaginalis Lb. reuteri Lb. reuteri Lb. thermotolerans
a b
Transformation frequency (per Ag pFX3 DNA) 99% (Acc. No. AY335495)a 99% (Acc. No. AJ421225) 99% (Acc. No. AY335495) 99.5% (Acc. No. AF257097) 98.5% (Acc. No. AF257097) 98% (Acc. No. AY339182) 99.3% (Acc. No. AF420311) 98.9% (Acc. No. AF420311) 99% (Acc. No. AF420311) 99% (Acc. No. AF420311) 99.5% (Acc. No. AF335475) 98.9% (Acc. No. AF335475) 100% (Acc. No. AY137589) 99% (Acc. No. AE017206) 99% (Acc. No. AE017206) 98% (Acc. No. AF243177) 99% (Acc. No. LF16SRR1) 98% (Acc. No. LR16SRR1) 98.8% (Acc. No. AF317702)
1.2102 Not detectedb 7102 Not detectedb Not detectedb 1102 3103 1.5103 2102 25 8.4104 10 Not detectedb 4103 4102 Not detectedb Not detectedb Not detectedb 10
Best alignment with 16S sequences from database. Less than 10 transformants per Ag pFX3 plasmid DNA.
Table 2 Electroporation of type strains of Lactobacilli using standard and modified protocolsa Species
Lactobacillus salivarius subsp. salivarius Lb. salivarius subsp. salivarius Lb. salivarius subsp. salicinius Lb. crispatus Lb. crispatus Lb. casei Lb. delbrueckii subsp. lactis Lb. Lb. Lb. Lb. Lb.
sakei curvatus curvatus acidophilus plantarum
Lb. delbrueckii subsp. bulgaricus Lb. helveticus a
Designation
Origin
Electroporation frequenciesb Standard protocol
Modified protocol
NCDO 8817
Turkey faeces
80
100
NCDO 929 NCDO 1555 NCIMB 702172 NCIMB 702240 ATCC 393 LK1 (Derivative of strain WS97) LTH 681 LTH 683 LTH 684 ATCC 4356 NCFB 1193
Not recorded Not recorded Infant faeces Human urine Dairy strain Not recorded
5103 1105 Not detectedc Not detected 4.5103 6106
3103 6103 Not detected 40 2104 5107
Raw sausage Raw sausage Raw sausage Human origin Ensiled vegetable matter Bulgarian yogurt Cheese starter culture
Not detected Not detected Not detected Not detected 2104
Not detected 10 Not detected Not detected 4105
Not detected Not detected
Not detected Not detected
NCDO 1489 CNRZ 32
For published electroporation data, see Chassy and Flickinger (1987, Lactobacillus casei); Zink et al. (1991, Lb. delbrueckii subsp. lactis); Schmidt et al. (1999, Lb. sakei); Klein et al. (1993, Lb. curvatus); Walker et al. (1996; Lb. acidophilus), Thompson and Collins (1996; Lb. plantarum); Bhowmik and Steele (1993; Lb. helveticus). b Transformants per Ag pFX3 plasmid DNA. c Less than 10 transformants per Ag pFX3 plasmid DNA.
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and Collins, 1996). Briefly, 12 ml volumes of cultured cells were resuspended in 1 ml of ice-cold ultrapure water and washed twice. Cells were then resuspended in 1 ml of 50 mM EDTA and held on ice for 5 min then washed once in ultrapure water and twice in 0.3M sucrose solution before being resuspended 100 Al of 0.3 M sucrose. The concentrated cell suspension (120) was electroporated using a Gene Pulser electroporator (Bio-Rad, Hemel Hempstead, UK) in cuvettes with a 0.2-cm electrode gap (Flowgen, Ashby de la Zouch, UK) with up to 5 Al plasmid DNA (at a concentration of 100 Ag/ml). Except where stated, electroporation parameters were 1.5 kV, 200 V parallel resistance, and 25 AF capacitance. For phenotypic expression, the cells were diluted immediately into 1.9 ml of MRS broth in a 2-ml vial pre-warmed to 37 8C. After 3-h incubation, serial dilutions were plated onto MRS agar containing the selective antibiotic (chloramphenicol or erythromycin, as appropriate). 2.4. Plasmids Plasmids used for electrotransformation experiments with Lactobacillus salivarius Sn1 are listed in Table 3. Experiments to determine optimum electroporation conditions for chicken crop isolates used plasmid pFX3. The functionality of erythromycin resistant genetic tools pG+host5 (Biswas et al., 1993) and pG+host 5:ISS1 (Maguin et al., 1996) was determined using Lb. salivarius strain Sn1 as host.
salivarius strain Sn1 (pG+host5:ISS1) grown in MRS broth containing erythromycin at 37 8C was diluted 1/100 into fresh medium without selection to allow exponential growth to resume (ca. 150 min). The culture was then shifted to the restrictive temperature (42 or 48 8C) for 150 min to reduce the plasmid copy number, then diluted and plated onto selective agar at the restrictive temperature (to detect transposition) and non-selective agar at the permissive temperature (to determine the viable count). Colonies from selective agar at the restrictive temperature were analysed for the presence of free plasmid and integrated pG+host5:ISS1. Total DNA was extracted following growth of the bacteria at 42 8C with selection and in the presence of glycine (0.13M) using Promega Wizard Genomic DNA (Promega, Southampton, UK) extraction kits according to the manufacturers’ instructions. Undigested DNA and DNA digested with HinDIII were run on 0.8% (w/v) agarose gels, blotted onto Hybond N+ membranes and probed with ISS1 PCR product, or linearised pG+host5 labelled using an Enhanced Chemiluminescence kit (ECL; Amersham, Little Chalfont, UK) according to the manufacturers’ instructions. General molecular biology procedures were as described by Sambrook et al. (1989).
3. Results 3.1. Identification of crop isolates
2.5. Generation of integrants using plasmid pG+ host5:ISS1 These experiments were essentially as described by Maguin et al. (1996). An overnight culture Lb.
The tentative identifications for strains of Lactobacilli used for electroporation experiments based on 16S rDNA sequence analysis are listed in Table 1.
Table 3 Plasmids used for the electroporation of Lb. salivarius Sn1 Plasmid
Origin
Selective antibiotic
Transformation frequencya
pFX3 pNZ123 pLP825 pIL253 pLP3537
pFX1 (Xu et al., 1991). Rolling Circle Replication. pWV01 (de Vos, 1987). Rolling Circle Replication. Lactobacillus plantarum plasmid p8014-2; (Posno et al., 1991). pAMg1 deletion derivative (Simon and Chopin, 1988). Theta replication. Lactobacillus pentosus plasmid p353-1; (Posno et al., 1991).
Chloramphenicol Chloramphenicol Chloramphenicol Erythromycin Erythromycin
2104 1107 2104 2104 6104
a
Transformants per Ag plasmid.
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3.2. Optimisation of the electroporation frequency using poultry isolates of Lactobacillus spp. as hosts During initial experiments, none of the 19 strains tested was electrotransfomable using the methods of Thompson and Collins (1996) or Walker et al. (1996). However, by adjusting the concentration of glycine in the growth medium over a range from 0.13 to 1.1M, a few transformants (b5) were obtained from two strains; Sn1 and A78. One of these strains (Lb. salivarius Sn1) was selected for further experiments. 3.3. A pulse of glycine rather than growth in glycine improves electroporation efficiency Measurements of optical density and determination of viable counts suggested that little or no growth of the bacteria ensued following dilution into fresh MRS broth containing 1.1M glycine (data not shown). Therefore, in order to increase the number of transformants, the optimum dilution factor from an overnight culture was determined over the range 1/20 to 1/2.5. Increasing the inoculum size enhanced the transformation frequency with a 1/6 dilution being the optimum. Shorter incubation times with the higher inoculum were then evaluated. The bacteria developed electrocompetence within 30 min following exposure to fresh medium containing glycine with the transformation frequency peaking at 60 to 120 min (Fig. 1). In the absence of glycine, electrocompetence still developed after 30-min incubation although there was a subsequent decline after 120-min incubation. This was not affected by the presence of sucrose (0.3M; data not shown). Using a 90-min pulse, the effect of increasing concentrations of glycine in the growth medium was measured. It appeared (Fig. 2) that more consistent transformation frequencies were obtained in the presence of glycine. A glycine concentration of 0.27M was selected for routine electroporation.
Fig. 1. Appearance of electrocompetence in Lb. salivarius strain Sn1 following incubation in fresh growth medium. An overnight culture of Lb. salivarius strain Sn1 was diluted 1+5 into fresh MRS containing 1.1 M glycine. Cells were harvested at intervals and electrocompetence determined. Data are taken from three experiments.
which the Lb. salivarius Sn1 cells were exposed. The absolute transformation frequency had not reached a maximum with the addition of 0.5 Ag pFX3 DNA, which was the highest quantity used. The electroporation efficiency determined in nine experiments using a range of DNA concentrations (0.1–500 ng/100 Al cell suspension) varied from 1.9103 to 1.6105 transformants per Ag pFX3 DNA (mean 1.7104). 3.5. Effect of host modification of transforming DNA Total plasmid DNA obtained from pFX3-containing transformants of Sn1 was used to electroporate strain Sn1 in order to determine whether the electroporation frequency could be enhanced by using plasmid DNA carrying the host strain modification. Total plasmid DNA was extracted from a pFX3 transformant of host strain Sn1 and the plasmid DNA was purified by isopycnic centrifugation. This DNA transformed strain Sn1 at a mean frequency of 1.6105 transformants per Ag which is a six-fold increase over the mean frequency obtained with unmodified pFX3 DNA.
3.4. Effect of increasing the concentration of transforming DNA
3.6. Transformation of crop isolates using modified protocol
There was a linear relationship between electroporation frequency and the DNA concentration to
Using the electroporation protocol adapted for crop isolates and, in separate experiments, pFX3
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one of the Lb. salivarius strains tested became electrocompetent. 3.7. Comparison of techniques for the electroporation of type cultures
Fig. 2. Effect of increasing glycine concentrations on electroporation efficiency of Lb. salivarius strain Sn1. An overnight culture was diluted 1+5 into fresh MRS broth containing increasing concentrations of glycine. Cells were harvested after a 90-min incubation and electrocompetence determined. Datapoints are taken from six experiments.
A number of type strains of different species of Lactobacilli (Table 2) were electroporated using two methods. First, the method of Thompson and Collins (1996) in which an arbitrary concentration of glycine (1%; 0.13M) was selected for growth. Second, the modifications described in the present investigation were used. When data from the two protocols were compared, the range of strains electrotransformable and the frequencies obtained were similar (Table 2). 3.8. Electroporation of Lb. salivarius Sn1 using a range of vectors
plasmid DNA or pFX3 plasmid DNA carrying potential Lb. salivarius strain Sn1 modification, the electrocompetence of previously non-transformable strains was determined (Table 1). A total of 11 out of the remaining 18 strains was electrotransformable using the adaptations to the technique and a maximum of 0.5 Ag of transforming DNA. All but
Electrotransformation frequencies obtained using additional plasmids are shown in Table 3. All four additional plasmids were able to transform the host strain. A relatively high transformation frequency was obtained using plasmid pNZ123. When poorly transformable Lb. curvatus strain LTH683 was electro-
Fig. 3. Temperature sensitivity of plasmids pG+host5 and pG+host5:ISS1 in host strain Lb. salivarius Sn1. The graphs show the retention of the erythromycin-resistance marker for (a) pG+host5 n and (b) pG+host5:ISS1 at 37 8C (open symbols) and 42 8C (closed symbols). Plate counts in the presence and absence of erythromycin were compared at intervals. Twenty-four-hour incubation represented approximately 24 generations at 37 8C and 40 generations at 42 8C. Data are means of three experiments.
.
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porated using plasmid pNZ123, transformants were obtained at a frequency of 1105/Ag using the modified protocol compared with a frequency of 6102/Ag using the standard protocol. 3.9. Transfer of genetic tools into strain Sn1 Plasmids pG+host5 and pG+host5:ISS1 were transformed into strain Sn1 and their presence confirmed by agarose gel electrophoresis and Southern hybridization. Differences in stability of the plasmids following growth at 37 and 42 8C were determined (Fig. 3). Both vectors were unstable when maintained at 42 8C compared with growth at 37 8C. Erythromycin-resistant colonies obtained following growth of strain Sn1 (pG+host5:ISS1) at 42 8C were purified and maintained at 42 8C in the presence of the selective antibiotic. Total DNA extracts from erythromycin-resistant colonies obtained at 428 were probed with ISS1. A total of 8 out of 14 colonies tested revealed the presence of plasmid DNA consistent with free pG+host5:ISS1 plasmid. The six remaining colonies yielded DNA which showed some evidence for the presence of free plasmid DNA but also an additional signal consistent with the presence of ISS1 on the chromosome (data not shown). The location of the ISS1 element from these putative integrants was determined by filter hybridization of ISS1 probe with HinDIII digested total DNA. DNA from one integrant gave weak signals with bands consistent with the ISS1 (or pG+host5) probes (data not shown). The data for DNA extracted from the five remaining colonies are presented in Fig. 4. These show intense signals predominantly with bands at 5.3 and 1.4 kb (vector and insert). In addition, the presence of two additional HinDIII fragments for each integrant is consistent with insertion of pG+host5:ISS1 at five independent sites. The experiment was repeated using selection at 48 8C which is the maximum growth temperature for Lb. salivarius Sn1. In this instance, putative integrants were obtained at a frequency of 0.02% and it was noted that these varied significantly in colony size. Of 10 isolates tested, none showed the presence of free plasmid. However, HinDIII digestion of their total DNA gave identical signals with the ISS1 probe. In addition to the 5.3- and 1.4-kb signals associated with
Fig. 4. Analysis of ISS1 integration in Lb. salivarius strain Sn1. Erythromycin resistant colonies of Lb. salivarius strain Sn1 (pG+host5:ISS1) following growth at 42 8C were recovered and purified. HinDIII digested total DNA was probed with ECL-labelled ISS1 DNA. The upper arrow indicates the position of linearised pG+host5 (5.3 kb). The lower arrow indicates the position of the fragment containing the ISS1 element (1.4 kb). The five lanes show the positions of the different patterns obtained.
the vector and insert, there were bands at 2.4 and 1.0 kb (data not shown).
4. Discussion 4.1. Electroporation of Lactobacilli from the chicken crop The modified electroporation protocol described in this report facilitated the electroporation of 12 out of 19 previously non- or poorly transformable strains of Lactobacilli isolated from the chicken crop. Six of seven strains of Lb. salivarius isolated from chicken crops were rendered electrocompetent or had their transformation frequencies improved following application of the modified protocol. By contrast, three type-cultures of Lb. salivarius were successfully electrotransformed by both the standard method and the modified protocol. One of these
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strains (NCDO1555) is the same as ATCC11742 which was successfully also electrotransformed by Heng et al. (1999). A total of three out of six strains identified as Lb. crispatus were transformed. One of two type cultures was electrotransformable but at a low frequency. In a previous report on the electrotransformation of Lactobacilli isolated from the chicken crop, Beasley et al. (2004) reported transformation of Lb. crispatus strains (two of five strains tested) at low frequencies (b20 per Ag) using a vector based on a cryptic Lb. crispatus plasmid. Frequencies obtained for some strains in the present investigation were an order of magnitude higher (100 to 700 per Ag). Of the remaining six strains, the isolates identified as Lb. johnsonii were only electrotransfomable using the modified protocol. This species has previously been described as being poorly transformable (Walker and Klaenhammer, 1994). The remaining strains studied belonged to species which have not been reported to become electrocompetent. Of the type of cultures which were amenable to electrotransformation, the technique described herein was in most cases at least as effective as growing the cells for extended periods in the presence of a cell-wall-weakening agent. Although we have not presented direct evidence for the presence of a modification of DNA passaged through Lb. salivarius Sn1, the observation that plasmid pFX3 extracted from Sn1 transformants was able to transform some of the target organisms more efficiently than unmodified pFX3 DNA would be consistent with this argument. Furthermore, the use of appropriately modified plasmid DNA may be a key factor for the successful transformation of strains with a functional restriction system. The protocol described in the present report contrasts with those described by, for example, Aukrust et al. (1995) who recommended mid-log cultures, Serror et al. (2002) who used early stationary phase cultures, and Zink et al. (1991) and Aymerich et al. (1993) who reported that some strains of Lb. delbrueckii subsp. lactis and Lb. sakei, respectively, gave highest electroporation frequencies on entering stationary phase. It was observed that all of the chicken crop isolates involved in the present investigation had a propensity to clump when incubated in MRS broth. When the bacteria were sub-cultured into
fresh medium, the period of incubation at which electroporation frequency was at its maximum, the clumps tended to disperse. A trivial explanation for this would be that more cells were available as targets for electrotransformation. Alternatively, cells entering exponential growth phase could be more susceptible to penetration by the DNA, and that the electrocompetence is maintained by the addition of glycine to the medium. The specific effect of glycine on these bacteria has not been identified although Hammes et al. (1973) suggested that glycine weakens the cell walls of Lactobacilli by reducing the number of cross links resulting in the disruption of the balance in peptidoglycan turnover and the misincorporation of glycine-containing precursors. Data from our laboratory and that of Beasley et al. (2004) suggest that bacteria from the chicken GI tract are recalcitrant with regard to electrotransformation. Some isolates can be rendered electrocompetent, however, by manipulating the growth conditions. Furthermore, the data suggest that empirical selection of a suitable plasmid for electrotransformation of a given strain will be an important consideration for the study of poorly transformable strains. A clear understanding of the nature of electrocompetence in poultry Lactobacilli will require further work. In addition, it appears that the requirement for optimisation of electroporation conditions is not only species and strain specific but may also have to be applied to bacteria isolated from any particular ecological niche. 4.2. Application of genetic tools to the genetic analysis of Lactobacilli from the chicken crop Plasmids exhibiting intrinsic temperature sensitivity when maintained in novel hosts have been used to deliver genetic markers to specific sites on the chromosome (Bhowmik and Steele, 1993; Serror et al., 2003). The intrinsic temperature sensitivity of certain plasmids has also permitted the development of novel vector systems for Lb. acidophilus and Lb. gasseri (Russell and Klaenhammer, 2001) and Lb. curvatus (Neu and Henrich, 2003). Plasmids pG+host5 and pG+host5:ISS1 are temperature sensitive derivatives of a Lactococcal plasmid pWV01-based replicon and exhibit marked instability in Lactococci (Biswas et al., 1993; Maguin
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et al., 1996) when the temperature is shifted from 28 to 37.5 8C. However, these genetic tools are generally not effective in Lactobacilli. For example, data from our laboratory (Steudel, 1996) showed that the vectors were stable in Lb. plantarum NCFB1193 following incubation at 28 or 37 8C and did not show instability until the growth temperature was raised to 41 8C when there was a 10-fold drop in the number of colony-forming units retaining the plasmid marker after a 20-h incubation. There was no evidence for random integration of the vector during these experiments. Gory et al. (2001) used a pG+host5-derivative to deliver a genetic material to a plasmid-cured strain of Lb. sakei but found that the plasmid was stable after 100 generations at 30 8C. It has been reported (personal communications in Serror et al., 2003) that the vector was not thermosensitive in either Lb. casei or Lb. plantarum. However, Russell and Klaenhammer (2001) noted that pGhost+5 precursor plasmid pGK12 was intrinsically temperature sensitive at 438 C in Lb. gasseri and Lb. johnsonii with only 0.01% of colony-forming units being retained after 30 generations at the elevated temperature. Pridmore (in Klaenhammer et al., 2002) reported that pG+host-based vectors were sufficiently temperature sensitive to be used efficiently for advanced genetic analysis in Lb. johnsonii. Because Lb. salivarius strain Sn1 appeared to have an optimum growth temperature of approximately 42 8C, and a maximum growth temperature of 48 8C, it was possible that the pG+host-based vectors could be functional in this host. This proved to be the case. The plasmid erythromycin resistance marker from pG+host5 and pG+host5:ISS1 was recovered in only 0.01% of colony forming units after a 6-h incubation (12 generations) at 42 8C. By contrast, at 37 8C the marker was still present in 50% and 10% of colony forming units after 30 generations for pG+host5 and pG+host5:ISS1, respectively. Although these data contrast with results obtained using Lactococci as hosts (Maguin et al., 1996) where the transposition frequencies of 10 6 and V10 2 for pG+host5 and pG+host5:ISS1, respectively, were reported, the thermal stability data are consistent with the findings of Serror et al. (2003) for transposition in Lb. delbrueckii subsp. bulgaricus at 44 8C when using related vector pG+host9. Although Serror et al. (2003) considered it unlikely that an ISS1-containing
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plasmid was actually transposing in Lb. delbrueckii subsp. bulgaricus, the same plasmid containing other lactic-acid-bacteria-derived insertion elements was able to transpose and into a range of chromosomal locations. Data presented here show that in Lb. salivarius plasmid, pG+host5:ISS1 can deliver the ISS1 element from the plasmid to a range of sites in the bacterial chromosome. It is probable that the integration has resulted in tandem duplication (Maguin et al., 1996). This is supported by the observation that in four of the five integrations (Fig. 4), the intensity of the bands for the pG+host 5 and ISS1 sequences using the relevant probes was greater than for the putative flanking HinDIII fragments. Single integrations of the genetic tool would be more desirable in order to prevent possible rearrangements and to facilitate sequencing of the flanking regions. In order to reduce the residual, free plasmid in the integrants, the selection temperature was raised to 488 C. Although this had the desired effect, the integrants tested appeared to be identical. This result was unexpected. It is possible that all colonies tested represent independent integrations into the same site but an alternative explanation is that the isolates were clonal and represent the most competitively growing integrants at the elevated temperature. Indeed the isolates selected for this experiment tended to be the larger colony-forming variants among the population. Further experimental work will therefore be necessary to optimise the conditions for selection of integrants. In conclusion, the modified electroporation protocol described and the temperature sensitivity of the genetic tool will permit gene expression analysis in this important group of organisms to be analysed and for knock-out mutants to be created for genes of interest.
Acknowledgements The authors wish to thank Mrs. Christine Nicholson and Mr. Gerard Cottrell for the expert technical assistance at different times during these experiments. One of us (CM) acknowledges the financial assistance from a DARDNI post-graduate studentship.
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