Appl Microbiol Biotechnol (1991) 35:334-338

017575989100140W Applied Microbiology

Biotechnology © Springer-Verlag 1991

Molecular cloning in Lactobacillus helveticus by plasmid pSA3::pVA797 co-integrate formation and conjugal transfer Keith Thompson ~ and Martin Collins 2

1 Food and Agricultural Microbiology Research Division, Department of Agriculture for Northern Ireland, and z The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, Northern Ireland, UK

Received 25 July 1990/Accepted 14 December 1990

Summary. A gene encoding fl-glucanase activity f rom Bacillus amyloliquefaciens was subcloned in both orien- tations into plasmid shuttle vector pSA3. In only one orientat ion could a co-integrate be generated with the conjugative plasmid pVA797. The plasmid co-integrate was conjugated into Lactobacillus helveticus strain CNRZ450, where it was stably maintained without an- tibiotic selection and exhibited fl-glucanase activity. This method of introducing cloned D N A into thermo- philic lactobacilli will facilitate the study of heterolo- gous gene expression in non-t ransformable species.

Introduction

In recent years there has been an upsurge in interest into the possibility of genetically manipulat ing the lac- ic-acid bacter ia (McKay and Baldwin 1990). Many spe- cies of lactic-acid bacter ia are now t ransformable and have thus become amenable to recombinant D N A tech- nology but there are as yet no generalized methods for t ransforming all strains, particularly those f rom ther- mophil ic dairy species such as Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus. Attempts to t ransform thermophil ic lactobacilli have been largely unsuccess- ful al though Batt (1986) and Boizet et al. (1988) have reported that low frequencies of t ransformat ion and transfection of L. delbrueckii ssp. bulgaricus protoplasts are possible. At present the only effective way of intro- ducing new D N A into these bacteria is by conjugation using broad-host- range conjugative plasmids such as pAMfll (Sasaki and Sasaki 1989 cited by Take tomo et al. 1989) and piP501 (Thompson and Collins 1988; Langella and Chopin 1989).

The cloning vector plasmid pSA3 (Dao and Ferretti 1985) can be introduced into strains of lactic-acid bac- teria by the generation of co-integrates with conjugative plasmid pVA797, a deleted derivative of piP501 (Evans

and Macrina 1983). This method had previously facili- tated the introduction of plasmid pSA3 into lactococci (Romero et al. 1987) L. plantarum (Shrago and Dobro- gosz 1988), and L. helveticus (Thompson and Collins 1989). The aim of the investigation described was to use the strategy of co-integrate format ion to clone a novel gene into L. heloeticus.

Materials and methods

Strains o f bacteria and conditions o f growth

The strains of bacteria used are listed in Table 1. Escherichia coli was cultured aerobically in Luria Bertani broth (Maniatis et al. 1982) at 37 ° C. Lactococcus lactis ssp. lactis was cultured in M17 broth (Oxoid CM785; Basingstoke, UK) containing 1% (w/v) glu- cose at 30 ° C, and Lactobacillus helveticus in MRS broth (Oxoid CM359) at 37 ° C. Both species of lactic-acid bacteria were cul- tured in a 5% (v/v) CO2 atmosphere.

All media were solidified with 1% (w/v) agar (Oxoid No. 1) when required. Filter matings were carried out as described by Thompson and Collins (1988).

Sources o f plasmid DNA

The plasmids used are listed in Table 2. An EcoRI fragment of DNA encoding a fl-glucanase gene from Bacillus arnyloliquefa- ciens cloned into plasmid pBR322 (plasmid pEG1) was a gift from R. Borriss (Berlin, Germany). Plasmids pSA3 and pVA797 were obtained from J. J. Ferretti (Oklahoma City, Okla., USA), and F. L. Macrina (Richmond, Va., USA), respectively.

Table 1. Strains of bacteria used

Species Strain Relevant designation phenotype

Escherichia coli Hbl01 Lactococcus lactis

ssp. lactis LM0230 Str r L. lactis ssp. lactis LM2345 Spc r Rif r Lactobacillus helveticus CNRZ450 str-501 Str r

Offprint requests to: J. K. Thompson Str, streptomycin; Spc, spectinomycin; Rif, rifampicin

Table 2, Plasmids used

Designation Relevant characteristics Source/reference

335

pEG1 fl-Glucanase gene from Bacillus amyloliquefaciens cloned into pBR322 Borriss et al. (1985) p S A 3 Streptococcus/Escherichia coli shuttle vector, Erm r Cmp r Tet r a Dao and Ferretti (1985) p V A 7 9 7 Conjugative; deletion derivative of piP501, Cmp r Evans and Macrina (1983)

pSA3b3 / fl-Glucanase gene from pEG1 subcloned into pSA3 in both orientations This investigation pSA3b6 J

Erm, erythromycin; Cmp, chloramphenicol a Tetracycline(Te0.resistance marker expressed in E. coli

Table 3. Concentrations of antibiotics used

Species Antibiotics (Ixg/ml)

Str Rif Spc Erm Cmp

Lactococcus lactis ssp. lactis 1000 20 200 5 10 Lactobacillus helveticus 250 2 5

Chemicals and reagents

DNA-modifying enzymes. Restriction endonuclease EcoRI and T4 DNA ligase were purchased from Amersham International (Bucks, UK) and used according to the manufacturer's instruc- tions.

Antibiotics. Erythromycin (Erm), chloramphenicol (Cmp), strepto- mycin (Str), rifampicin (Rif) and spectinomycin (Spc) were pur- chased from Sigma (St. Louis, Mo., USA). The concentration of antibiotic incorporated the media differed according to the host species (Table 3).

fl-Glucanase detection. The method of Cantwell and McConnell (1983) was used. The lichenan substrate and Congo Red indicator for the detection offl-glucanase activity were purchased from Sig- ma.

DNA hybridization. An ECL Gene Detection kit (Amersham Inter- national) was used for the detection of homologous DNA se- quences. The methods for preparation of gels, transfer of DNA onto nitrocellulose membranes and the detection of labelled DNA used protocols supplied by the manufacturer.

Molecular cloning procedures

Sub-clones of the fl-glucanase gene in pSA3 were identified by the insertional inactivation of the crop r marker and by the production offl-glucanase activity. Confirmation of the presence of the fl-glu- canase gene was made using a probe consisting of EcoRI linear- ized pEGI DNA.

Plasmid analyses

Plasmid DNA in Laetocoecus lactis ssp. lactis derivatives was de- tected by the method of Anderson and McKay (1983) and in Lac- tobacillus helveticus by the method of Klaenhammer (1984).

Results

Subcloning o f the fl-glucanase gene and co-integrate formation

The 3.6-kb EcoRI fragment from pEG1 was subcloned into pSA3 in both orientations to give plasmids pSA3b3 and pSA3b6 (Fig. 1). The plasmids were transformed separately into Lactococcus lactis ssp. lactis strain LM2345 and the conjugative plasmid pVA797 was in- troduced by filter matings. The resultant strains (LM2345 [pSA3b3, pVA797] and LM2345 [pSA3b6, pVA797]) were used to construct co-integrates. Trans- conjugation frequencies are shown in Table 4. Simulta- neous transfer of erm r and cmp r markers was observed only with strain LM2345 [pSA3b6, pVA797] as donor.

Putative co-integrate-containing transconjugants harboured a single plasmid with an approximate size o f 44 kb, which hybridized to the fl-glu gene probe (Fig. 2). When this putative co-integrate-containing strain was used as a donor in conjugation with plasmid-free lacto- cocci, the erm r and cmp r markers transferred at a fre- quency of 2.5 x 10 -a /donor bacterium (Table 4). By contrast, repeated attempts to generate co-integrates between plasmids pSA3b3 and pVA797 were not suc- cessful.

Conjugal transfer o f the plasmid co-integrate to Lactobacillus helveticus strain CNRZ450

Following filter matings between Lactococcus lactis ssp. lactis strain LM2345 [pSA3b6: :pVA797] and Lactoba- cillus helveticus strain CNRZ450 str-501, transconjugant

~ R I ~ o R I

~ "EcoR i ~ ~ ~ I ~ E c o R I

Aval Aval Fig. 1. Diagrammatic representation of the structure of plasmids pSA3b3 and pSA3b6 showing the relative positions of the EcoRI and AvaI sites, and the position of the fl-glucanase determinant (labelled fl-glu)

336

Table 4. Conjugation and co-integrate for- mation in Lactococcus lactis ssp. lactis strains

Donor strain Recipient M a r k e r s Transconjugation strain selected frequency a

LM2345[pSA3, pVA797] LM2345[pSA3b3, pVA797] LM2345[pSA3b6, pVA797] LM0230[pSA3b6: : pVA797]

LM0230 str rerm r c rop r 10 - 4

LM0230 str ~ erm r crop r < 10-8 LM0230 str r ermr cmp r 10 -7 LM2345 rift spc ~ erm ~ cmp r 2.5 × 10-4

" Per donor bacterium

Fig. 2 A, B. Analysis of co-integrate-containing Lactobacillus hel- veticus strain CNRZ450 derivative. Lysates of an Erm r Cmp r L. helveticus strain CNRZ450 transconjugant (lane 2) and Lactococ- cus lactis ssp. lactis strain LM2345 [pSA3b6: :pVA797] (lane 3) were run on an agarose gel and the plasmid mobilities compared (A). The DNA from the gel was then transferred to a filter by Southern blotting and hybridized to a labelled fl-glucanase gene probe (B). Lane 1 in both figures shows plasmid pSA3b6. The sizes of the plasmids arrowed are in kilobases. Additional bands in lanes 1 and 3 are almost certainly multimeric forms of the plas- mids in combination with open circular and linear forms. The sen- sitivity of the enhanced chemiluminescence gene detection system enables these additional bands to be visualised in B

clones were detected at a frequency of 10 -7. These clones were able to form turbid cultures in MRS broth containing up to 100 Bxg ml-1 of erythromycin and had simultaneously acquired the unselected c m p r marker. Lysates of ErmrCmp r transconjugants of L. helvet icus CNRZ450 revealed a novel band of similar mobility to plasmid pSA3b6: :pVA797 from L a c t o c o c c u s lactis ssp. lactis strain LM2345 (Fig. 2). This band also gave a hy- bridization signal when probed with the labelled f l-glu gene.

Express ion o f f l -g lucanase act iv i ty

When MRS agar containing lichenan was inoculated with co-integrate-containing clones and incubated at 37°C for 48 h, a zone of hydrolysis of the substrate could be detected (Fig. 3). By contrast, no activity was

Fig. 3. Hydrolysis of lichenan by Lactobacillus helveticus strain CNRZ450 derivatives harbouring plasmid pSA3b6::pVA797 co- integrates. An MRS agar medium containing 0.2% (w/v) lichenan was spot-inoculated with L. helveticus strain CNRZ450 [pSA3::pVA797] and two derivatives (Col and Co2) that har- boured the fl-glucanase gene in a putative pSA3b6::pVA797 co- integrate. After incubation at 37 ° C for 48 h in an atmosphere con- taining 5% COz the plates were flooded with a 0.1% (w/v) aque- ous solution of Congo Red. Zones of clearing, indicating that the substrate had been hydrolysed, became visible after up to 120 min

observed when the Lac tococcus donor strain contained either pSA3b6 or the pSA3b6::pVA797 co-integrate. Both these plasmids, however, showed a high level of fl-glucanase activity in E. coli with zones of hydrolysis of lichenan of 6-7 mm radius.

Stab i l i t y o f c loned f l -g lucanase gene in Lac tobac i l lus helvet icus strain C N R Z 4 5 0 growing wi thou t select ion

A culture of L. heivet icus strain CNRZ450 [pSA3b6: : pVA797] growing in the presence of selective antibiotics was spread-plated onto MRS agar contain- ing lichenan. A total of 59 out of 60 colonies that grew were still able to hydrolyse lichenan.

A culture was then serially transferred into fresh an- tibiotic-free MRS broth at 24-h intervals and the pro- portion of antibiotic-resistant or fl-glucanase-negative clones determined. After four serial daily transfers, al- most 100% of clones were antibiotic resistant and exhi- bited fl-glucanase activity. There was no significant loss of either marker until the seventh transfer when ap- proximately 25% of colonies recovered from non-selec- tive agar retained both the markers.

337

Discussion

Evidence for the transfer o f the plasmid pSA3b6: :p VA 797 co-integrate into L. helveticus

The physical and genetic evidence presented are consis- tent with the transfer of the fl-glucanase gene from B. amyloliquefaciens to L. helveticus via plasmid co-inte- grate formation and conjugal transfer. Transfer of the plasmid pSA3b6::pVA797 co-integrate to L. helveticus CNRZ450 was shown to occur at a low but detectable frequency (ca. 10-7). Cloned DNA can therefore be in- troduced into the new host if it is capable of being in- corporated into a plasmid co-integrate. The strategy therefore depends on the in-vivo generation of the plas- mid co-integrate. This event did not occur when the fl- glucanase gene was in the opposite orientation (plas- mid pSA3b3). In further experiments the phospho-fl- galactosidase gene cluster from L. casei strain 64H (Lee et al. 1982) was cloned in both orientations into a PstI site on plasmid pSA3 at map position 6800 bp from the AvaI start point. Although co-transfer of the erm ~ and cmp r markers between lactococci could be detected, in neither orientation was a stable co-integrate with plas- mid pVA797 detected (data not shown).

The precise method by which co-integrates are formed is uncertain. Romero et al. (1987) postulated that the plasmids might undergo recombination be- tween homologous tracts of DNA. By contrast, recent experiments on the mobilization of streptococcal plas- mid pMV158 have shown that this non-conjugative plasmid encodes features which facilitate its own mo- bilization (Priebe and Lacks 1989; van der Lelie et al. 1990). These include a plasmid recombination enzyme and the palindromic sequences important as targets for the enzyme.

We found that in pSA3b6::pVA797 co-integrates, the plasmid pSA3b6 moiety resides on the 12-kb BstEII fragment of plasmid pVA797 (data not shown). The 2- kb region of homology between pSA3 and pVA797 (Romero et al. 1987) is harboured within the 6.5-kb BstEII fragment of pVA797 (Evans and Macrina 1983). This result is not, therefore, consistent with co-integrate formation having arisen as a result of homologous re- combination between the plasmids. Thus it is possible that failure to form co-integrates could be due to non- functioning of either the putative recombinational en- zyme for co-integrate formation, or the target sequence, when the fl-glucanase gene has been cloned in the orientation in pSA3b3.

Expression o f the fl-glucanase gene

fl-Glucanase activity was detected in the presence of pVA797::pSA3b6 plasmid co-integrates in E. coli and L. helveticus but not Lactococcus lactis ssp. lactis. By contrast, plasmid pSA3b3 directed lichenan hydrolysis in L. lactis ssp. lactis. There must therefore be a feature of the region of DNA that controls the expression of the fl-glucanase gene in the pSA3b6 orientation that al-

lows a high level of expression in E. coli but prevents the initiation of, or terminates the transcription of, the gene in L. lactis ssp. lactis. It is known from sequencing data that gene expression signals differ in Gram-posi- tive and Gram-negative bacteria and between for exam- ple lactic streptococci and B. subtilis (see review by de Vos 1987) but these structures or signals in pSA3b6 ap- pear to be recognized in Lactobacillus helveticus.

Stability o f the cloned DNA in L. helveticus strain CNRZ450 [pSA3b6: :pVA797] growing in batch culture without antibiotic selection

The L. helveticus CNRZ450 [pSA3b6: :pVA797] clone appeared to be maintained stably without antibiotic se- lection for 96 h (four transfers, up to 40 generations). This result contrasts with our earlier data (Thompson and Collins 1989), which showed that plasmid pSA3::pVA797 co-integrates were stable during sub- culture without selection for 48 h only (ca. 20 genera- tions). Bates et al. (1989) found that in L. plantarum a celE gene from Clostridium thermocellum cloned into pSA3 was relatively unstable. Only 10% of cells re- tained the erm r marker after 45 generations. In similar experiments, Scheirlinck et al. (1989) observed that 5% of cells retained a recombinant pSA3 plasmid after 26 generations. It is possible, however, that the cloned fl- glucanase gene confers a selective advantage to the bac- teria growing in MRS media.

In conclusion, the protocol described may have some application for the study of heterologous gene ex- pression in lactic-acid bacteria that are not transforma- ble. Although limited by the inability of certain cloned DNA sequences to generate co-integrates with conjuga- rive plasmids, the fact that these plasmids are stably re- plicated, in contrast to those that replicate by the roll- ing circle method (Swinfield et al. 1990), makes them an attractive vehicle for the construction of recombinant molecules in lactobacilli. Problems associated with co- integrate generation may well be overcome when the molecular mechanisms at work are fully understood.

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