In vivo gene transfer systems and transposons

Biochimie 70 (1988) 489-502 (~) Soci6t6 de Chimie biologique/Elsevier, Paris

489

In vivo gene transfer systems and transposons Gerald F. FITZGERALD 1 and Michael J. GASSON2

1Department of Food Microbiology, University College, Cork, Ireland, and 2AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich, U.K. (Received 15-12-1987, accepted 23-12-1987)

Summary - -

The continued development of our understanding and application of the in vivo gene transfer systems, transduction and conjugation, and the more recent use of transposons in lactic acid bacteria is reviewed. The discovery of transduction and its use as a tool for genetic analysis is described. The history of the use of conjugation in lactic acid bacteria is outlined, but more detailed discussion is reserved for in-depth analysis of the conjugation system associated with the lactose plasmids of Streptococcus lactis strains ML3 and C2. This system is notable for an unusual cell aggregation phenotype associated with variants of lactose plasmids capable of high frequency transfer and the complex DNA interactions associated with this property. Recent advances in the use of wide host range conjugation systems, such as that of plasmid pAM/31 are described, including the mobilisation of vectors by cointegrate formation and subsequent segregation after transfer. The successful exploitation of conjugation for the construction of bacteriophage-resistant starter cultures is highlighted. A description of transposable genetic elements in the lactic acid bacteria, both insertion sequences and transposons, puts emphasis on the elegant analysis of insertion sequence ISL1 in LactobaciUus casei and on the exploitation of the conjugative transposons Tn916 and Tn919. The latter is especially important for providing a technology to initiate analysis of the bacterial chromosome of the lactic acid bacteria. •--+w..+ o+xu

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Introduction The widespread use of the lactic acid bacteria for biotechnological processes, particularly dairy and other food fermentations, has undoubtedly provided a major stimulus for recent advances in the genetic analysis of these bacteria. The availability of gene transfer systems, particularly the in vi:,o systems of transduction and conjugation, has been an important factor in the study of a number of key areas, such as the role and structure of plasmid DNA. Both conjugation and transduction have been used as manipulative techniques to demonstrate that most of the economically important traits of these bacteria (e.g., lactose, citrate, protein utilisation, bacteriophage resistance) are plasmid coded. Transduction has also been especially useful in the structural analysis and mapping of plasmid mole-

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cules from lactic streptococci. As well as describing these processes and referring to the markers transferred, we discuss some of their more interesting aspects, including the stabilisation of transduced markers by integration into the recipient chromosome and cointegrate formation associated with the clumping phenomenon described in some conjugation systems. The molecular basis of some of these events is currently being elucidated and details of the better studied systems are described. The mounting evidence that transposable elements are mediating many of these processes is reviewed. In the final section we will also review the recent developments regarding the application of transposon technology to the lactic streptococci in which transposons of the Tn916 family have been used to target genes, particularly those of chromosomal origin.

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G. F. Fitzgerald and M. J. Gasson

Transduction Transduction is a method of gene transfer mediated by bacteriophage. The phenomenon was first described by Zinder and Lederberg [1] in Salmonella when phage P22 was shown to transfer amino acid independence to auxotrophic mutants. This type of genetic exchange was termed generalised transduction, since any marker had a more or less equal probability of being transferred. Soon afterwards, specialised or restricted transduction was reported in E. coli [2]. In this case, only genes adjacent to the inserted prophage on the E. coli chromosome (i.e., gal and bio) were transduced as a result of erroneous excision of the prophage DNA. Subsequently, transduction has been described in many other bacterial genera, which is not surprising, considering the widespread occurrence of lysogeny in bacteria. In the case of the lactic acid bacteria used in food fermentations, transduction, with one exception, has been reported only in the lactic or Group N streptococci. This probably reflects very limited attempts to demonstrate the phenomenon in the other bacterial groups involved, i.e., Lactobacillus, Leuconostoc, Streptococcus thermophilus and Pediococcus. Recently Mercenier et al. [3] showed that two virulent bacteriophages of S. thermophilus were able to transduce a number of different sized plasmid molecules. T--he earliest reports of transduction in the lactic streptococci came a mere decade after its initial discovery in enteric bacteria when the transfer of tryptophan independence and streptomycin resistance to S. lactis ssp. diacetylactis 18-16 and S. lactis C2, respectively, using virulent phage, was described [4, 5]. However, there were no further reports of gene transfer of any type in the lactic streptococci for the next ten years until a series of papers from McKay's laboratory described the development of a transduction system for S. lactis C2 using temperate phage. In 1973, McKay et al. [6] reported the transduction of Lac into a Lac- derivative of S. lactis C2, using a UV-induced phage lysate from the parental culture. The non-availability of indicator strains or prophage cured derivatives of strain C2 prevented the enumeration of phage in the lysate, but the number of transductants obtained was proportional to the amount of lysate used. The phenomenon of high frequency transduction (HFT) previously described in E. coil [7] was also described. In this instance, a

UV-induced lysate prepared from a Lac ÷ transductant of S. lactis C2 had about 100 times the transducing ability of the original lysate. Furthermore, the Lac ÷ character was particularly unstable in the HFT transductants supporting the accumulating evidence of that time that Lac was a plasmid coded trait. In contrast, transductants into which the maltose and mannose markers had been transferred were incapable of yielding HFT lysates. Also, both traits were stably maintained, suggesting a chromosomal location for these genes. The intermittent co-transduction of Prt with Lac in S. lactis C2 has also been described even though Prt was an unselected marker in the transfer experiments [8]. This, taken together with the reported spontaneous and simultaneous loss of both characters in some strains provided early evidence that Lac and Prt are closely associated and often reside on a single plasmid. However, when Molskness et al. [9] transduced Lac, also into a Lac- S. iactis C2 recipient, cotransduction of Prt could not be demonstrated. The Prt status of Lac + transductants of S. lactis C2 derived using mitomycin C induced prophage from S. lactis C20 was not determined [10]. While the demonstration of transduction as a gene transfer system in any bacterial genus does have its merits, its greatest value lies in its use as a tool for genetic analysis. This has certainly been true in the case of the enteric bacteria and it is also true, although to a lesser extent, in the lactic streptococci. Transduction hfis been exploited to confirm the plasmid-associated nature of Lac in S. lactis C2 [11]. Although the analytical techniques of the time were relatively cumbersome (i.e., analysis of satellite peaks from 3H-labelled cell lysates following CsCI density gradient centrifugation and contour length measurements of plasmid molecules by electron microscopy), clear evidence was presented that Lac ÷ Prt ÷ and Lac ÷ Prt- transductants of S. lactis C2 harboured a plasmid of approximately 20-21 MDa in size which could not be detected in Lac- derivatives isolated either spontaneously or after acriflavine treatment. However, since similar curing data had linked Lac to a 30 MDa plasmid in the parental S. lactis C2, the question arose as to the origin of the 20-21 MDa molecule in Lac ÷ transductants. The likely answer was provided when Klaenhammer and McKay [12] isolated two defective transducing phages from S. lactis C2 following UV irradiation .and demonstrated that each had slightly different head sizes that

Gene transfer systems and transposons

were capable of accommodating 22.6 and 23.8 MDa of phage DNA, respectively. Based on this information McKay et al. [11] speculated that the limiting size of the phage head resulted in a process termed transductional shortening, whereby only deleted derivatives of the 30 MDa Lac plasmid could be accommodated, and thereby accounting for the observation of the 20-21 MDa molecules in Lac ÷ transductants. Thus, deleted derivatives of the Lac plasmid (or presumably any other plasmid) could be incorporated into transducing phage heads provided they fell within the maximum and minimum size limits for packaging and these limits will be determined by the size of the native phage DNA. This conclusion also implied that transducing phage contained no phage DNA. It is also noteworthy that both of the phage types isolated by Klaenhammer and McKay [12] were equally capable of transducing Lac, Prt and maltose markers, although the HFT phenomenon was observed to occur with only one of the phages. However, the reason and the significance of this latter finding were not explained. While most of the early work on characterisation and development of transduction systems in the lactic streptococci was confined to S. lactis C2, later reports by Gassor, and his colleagues described an apparently similar transduction process in the closely related S. lactis 712. The phenomena of co-transduction of Prt with Lac, transductional shortening and HFT have been described for strain 712 and a molecular analysis of these events has helped provide an explanation for these observations [13-17]. Using restriction enzyme analysis, Gasson and Warner [18] demonstrated that the transductionally shortened plasmids observed in Lac ÷ Prt ÷ and Lac ÷ Prt- transductants contained no phage DNA and were, in fact, deletion derivatives of the native L a c / P r t plasmid (pLP712) of the parental S. lactis 712. Thus, while Klaenhammer and McKay [12] had previously proposed that the HFT phenomenon observed in S. lactis C2 may have involved recombination between transducing phage and Lac plasmid DNA, the restriction data suggest that this is not the case in the S. lactis 712 transduction system at least. Gasson and Davies [17] have proposed that the transductional shortening phenomenon in S. lactis 712 arises from the preferential packaging of preformed spontaneously deleted derivatives of pLP712 and they have provided experimental evidence that HFT depends solely on the availability of a transductionally shortened plasmid

491

[19]. Consequently, HFT can be explained on the basis of first generation transductants all having a Lac plasmid of the appropriate size to be accommodated in the transducing phage head and thus lysates induced from these transductants will have a higher probability of packaging the shortened plasmid DNA. The observed intermittent co-transduction of Pr~ with Lac can also be explained by this model. Lac ÷ Prt ÷ transductants harboured plasmids all of which were identical on the basis of restriction mapping and contained a single deletion [18, 17]. The freo'lent occurrence of this same deletion implies a preferential event within the overall inherent instability of pLP712 [14, 20]. In contrast, all Lac ÷ Prt- transductants harboured plasmids with varied multiple deletions. This observation was explained by the relative location of prt, lac and replicator genes on plasmid pLP712. A single deletion that removed prt genes but left lac and replicator genes intact would be too small to reduce the size of pLP712 sufficiently for accommodation in the bacteriophage head. This has been borne out in restriction analysis of plasmids from Lac ÷ Prt- transductants [18]. It is also noteworthy that transduction of the MLS resistance S. faecalis plasmid pAM/31 to S. lactis 712 has been described by Davies and Gasson [19]. However, the HFT phenomenon typical of Lac plasmid transfer could not be detected in repeated transduction experiments. This may be explained by the fact that pAM/31, at 17 MDa is already small enough for packaging in prophage heads and elevated transduction by deletion is not possible. Recently, efficient transducfion of small vector plasmids, such as pCK1, hag also been demonstrated, su[;gesting that a minimum size limit for transduction does not exist (P. Anderson and M. Gasson, unpublished data). A striking feature of most strains of lactic streptococci is the plasmid coded nature of many key functions important in the context of using these bacteria in industrial fermentations (see Gasson and Davies [ 17] and Kondo and McKay [21] for reviews). The consequence of this is that many desirable properties, such as lactose, protein and citrate utilisation, are unstable. Obviously, the stabilisation of traits such as these by integration of the appropriate genes in the chromosome :s highly desirable. In this context, the observation of McKay and Baldwin [22] describing the stabilisation of Lac and Prt markers in S. lactis C2 transductants is of major significance. These transductants (three of which

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G. F. Fitzgerald and M. J. Gasson

were Lac ÷ Prt- and one Lac * Prt ÷) contained no detectable plasmid DNA and retained their phenotypes in continuous culture and following growth in the presence of acriflavine, a plasmid curing agent, data which suggested that the l a c / p r t genes had integrated into the recipient chromosome. The chromosomal location of the lac genes was confirmed when this marker was transduced at frequencies typical of a chromosoreally rather than plasmid located gene following irradiation of a transducing lysate to low doses of UV light in the manner described by Arber [23]. A further observation was that the level of gene expression was reduced in the stabilised transductants possibly due to a gene dosage effect. Interestingly, Kempler et al. [24] showed that the stabilised Lac + Prt* transductant of S. lactis C2 which was less proteolytic than the wild type was capable of producing a superior Cheddar cheese with reduced bitterness levels. Although the stabilisation phenomenon has also been described in the S. lactis 712 and S. lact/s ML3 (using S. cremoris C3 iysogenic transducing phage in the latter case) transduction systems [ 19, 25], the molecular basis of these events has not yet been established. For example, it would be interesting to know if the integration process is dependent on host recombination (Rec) functions and the availability of a Recderivative of S. lactis ML3 should make such a study feasible [26]. In fact, transduction has K.lll.,Ik.,il llll I.lll~:; Re C ~ bUtbd-l ~, ' .- " . . of . [II~l ~". . . . . .U~l~Ik.l . ~ "lk~l¢ . . .~.O l l l l'~--"'-~ mutants. The basis of these experiments lies in the fact that an intact host Rec system is required for the integration and expression of chromosomal markers introduced by transduction, whereas plasmid coded traits do not require integration for expression and therefore, these can be successfully introduced into a Rec deficient strain. Anderson and McKay [26] isolated a Recderivative of S. lactis ML3 based on its increased sensitivity to methyl methanesulfonate (MMS) and UV light. The Rec- nature of the mutant was confirmed when transduction of the chromosomal streptomycin resistance (Str) marker could not be demonstrated, while Lac plasmid transfer did occur. A limiting factor relating to the exploitation of transduction as a tool for the genetic analysis of lactic streptococci is the specificity of the transducing phage for its own host. Nevertheless, Davies et al. [27] have demonstrated the ability of the temperate phage from S. lactis 712 to transduce lac genes into Lac- derivative of S.

lactis strains C2 and ML3. However, these strains are closely related and are ultimately derived from a single source [27]. Significantly, these authors were unable to demonstrate transduction to other unrelated S. lactis strains using the same transducing lysate. In addition, McKay et al. [6] were unable to show transduction of Lac to S. cremoris and S. lactis ssp. diacetylactis strains using an S. lactis C2 transducing lysate. However, interspecies transduction in the lactic streptococci was reported by Snook et al. [25] when a mitomycin C induced prophage from S. cremoris C3 was successfully used to transduce Lac into Lac- derivatives of S. lactis C2 and ML3. Transduction occurred at a very low frequency and co-transduction of the Prt marker was not observed. Most of the transductants characterised possessed transductionally shortened Lac plasmids but no plasmid DNA could be detected in a small number of transductants, suggesting chromosomal integration of the lac genes. Transduction is one of a number of gene transfer systems operating in the lactic streptococci and while it may not be as relevant as other mechanisms, like conjugation or protoplast transformation, it has made a significant contribution to the advancement of genetic studies of these bacteria. A number of aspects of the transduction process, such as the host specificity of transducing phage and the stabilisation of plasmid coaea trmts, warrant further investigation. The integration of plasmid coded genes into the chromosome of some transductants also has obvious commercial applications. However, in other bacterial systems, particularly enteric bacteria, transduction has been used to greatest advantage in the construction of chromosomal linkage maps, an application that is also very desirable in lactic streptococci. While the small capacity of the phage head may limit the use of transduction in this context, a more serious problem is the paucity of known genetic markers on the chromosome of these bacteria. Thus, the recent application of transposon technology to the lactic streptococci to target chromosomally located genes represents an important advance in the genetic analysis of these bacteria. These developments and the role of the transposable elements in the lactic acid bacteria in general will be reviewed later.

Conjugation Gene transfer by bacterial mating or conjugation

Gene transfer systems and transposons is a widely described phenomenon in the lactic acid bacteria. Earliest reports are of conjugal transfer of lactose genes [28, 29] and of the introduction into lactic streptococci of the antibiotic resistance plasmid pAPA/31 from a hospital isolate of Streptococcus faecalis [30]. Much of the subsequent literature is concerned with cataloguing further instances of conjugation involving these phenotypes. In addition to numerous reports involving S. lactis and S. cremoris [31-38], lactose plasmid transfer has been described in Lactobacillus casei [39]. Only one conjugation system, that associated with the lactose plasmids of S. lactis strains ML3 and 712 [31, 54-57], has been subject to a detailed analysis and in this case a complex and intriguing mechanism involving transposition of DNA has been described. The molecular structures of the introduced wide host range plasmid pAM/31 and some similar plasmids have been reported and genetic determinants for their transfer systems have been located on restriction maps [39, 40]. The most significant recent advance in the use of such wide host range plasmids has been their mobilisation of plasmid cloning vectors, thereby enabling strains that have proved difficult to transform directly to feature in genetic engineering experiments [41, 42]. Conjugal transfer of other plasmid-associated phenotypes has included bacteriocin production [42-45], sucrose fermenting ability and nisin l.,S,L, u u v = . - u s ,

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and bacteriophage resistance [35, 37, 50-53]. At the level of industrial application, conjugation has been used to select a nisin-producing strain with dramatically enhanced yield [49] and also for the construction of bacteriophage-resistant dairy starter strains [53]. In the latter case, the constructs have been successfully used by the dairy industry. Lactose plasmid conjugal transfer is generally a rather inefficient process, with the notable exception of certain plasmids created as a direct consequence of a mating experiment. When Streptococcus lactis 712 was used as a donor in mating experiments with a Lac- derivative as recipient, the rare progeny colonies that were isolated included variant types with an unusual morphology. Broth cultures grown from these variant colonies exhibited strong cell aggregation or clumping and when they were used as donors in subsequent conjugation experiments, the transfer frequei~cy was found to be highly efficient. The assumption that this change was caused by derepression of a transfer system led

493

Gasson and Davies [31] to refer to aggregating cultures as Lax-, implying that the phenotype was controlled by a gene lax (lax = loose, not compact). This simple explanation has subsequently proved to be incorrect and the origin of aggregating progeny has been shown to involve a complex transposition process in which a transfer factor encoded by another plasmid becomes cointegrated with the lactose plasmid. The molecular events involved in the generation of aggregating progeny strains began to be revealed by Walsh and McKay [54] (who made similar observations of progeny colonies that had aggregation phenotypes and enhanced transferability) when the lactose plasmid of S. lactis ML3 was transferred into S. lactis LM2301, a plasmid-free derivative of S. lactis C2. Analysis of plasmids introduced into S. lactis LM2301 revealed the presence of a 104 kb lactose plasmid that was not to be found in the donor strain, S. lactis ML3. The lactose plasmid pSK08 that was present in the donor strain was a molecule of only 55 kb and Walsh and McKay [54] suggested that the genes responsible for cell aggregation and high frequency conjugation were on a segment of DNA that recombined with the 55 kb lactose plasmid in S. lactis ML3. Walsh and McKay [55] used restriction e.ndonuclease digestion to show that the 104 kb plasmid from an aggregating progeny strain was not a simple dimer of the lactose plasmid pSK08, but that it ~,~,/11 ¢¢lllll~gl

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quently, Anderson and McKay [56] made a detailed analysis of 15 enlarged lactose plasmids taken from independently isolated transconjugants. These included both transconjugants which exhibited cell aggregation and high transfer frequency, but also examples in which cell aggregation a n d / o r high transfer frequency did not occur. The presence of enlarged plasmids in strains of the latter type had already been observed by Walsh and McKay [55]. Anderson and McKay [56] observed that an additional plasmid of 48 kb was present in the donor strain, S. lactis ML3, and that this plasmid, pRS01, constituted the foreign DNA that was introduced into enlarged lactose plasmids following their conjugal transfer. A restriction digestion analysis of plasmid pRS01, the lactose plasmid pSK08 and enlarged plasmids from the fifteen independently isolated transconjugants provided details of the plasmid cointegration events that had occurred. In all but one case, it was shown that the cointegration event involved a specific region of plasmid pSK08 but that the

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G. F. Fitzgerald and M. J. Gasson

cointegration site on pRS01 varied widely. Five distinct regions of cointegration on pRS01 were identified and the respective enlarged plasmids were shown to confer different properties on their host cells. One class of plasmid cointegrates inhibited cell aggregation without high frequency transferability and another group, which did not have cell aggregation, transferred at an intermediate frequency. This information allowed genetic regions that were involved in cell aggregation and transfer to be located on the restriction map of pRS01 and provided the beginnings of a characterisation of the functions encoded by pRS01. Anderson and McKay [56] made another important observation with respect to cointegrate formation. A recombination-deficient derivative of S. lactis ML3 had earlier been isolated by Anderson and McKay [26] and this was used to show that conjugal transfer and the creation of cell aggregation and high transfer frequency phenotypes associated with plasmid cointegration s'ill occurred in the absence of a generalised recombination system. Analysis of the restriction endonuclease fragments of pRS01 and pSK08 that were directly involved in cointegrate formation provided another important observation. When these fragments were compared with the equivalent fragments taken from seven different plasmid cointegrates, it was consistently observed that the cointegrate fragments were enlarged by approximately i kb. This observation and the Rec-independent nature of the event led Anderson and McKay [56] to postulate that cointegration was caused by a transposition event probably involving an insertion sequence of about 1 kb in size. This concept has recently been proven with the isolation and DNA sequence determination of the suspected insertion sequence. Polzin and Shimizu-Kadota [57] examined the junction regions of the cointegrate plasmid pPW2 as well as the corresponding regions of the lactose plasmid pSK08 and the transfer factor encoding plasmid pRS01. On pSK08 a new insertion sequence named ISS1S was discovered which is involved in and duplicated during the formation of pPW2. The insertion sequence ISS1S was 808 bp in size and had 18 bp inverted terminal repeats with the sequence GGTI'CTGTGCAAAGTTT. The DNA sequence included an open reading frame that would encode a putative protein of 266 amino acids and transposition generated an 8 bp direct repeat in the target DNA. The DNA sequence of ISS1S

showed strong homology with the Gramnegative transposon IS26. Analysis of plasmids involved in S. lactis 712 lactose gene transfer has also produced interesting data. In this strain, the lactose plasmid is a 55 kb molecule pLP712 that also encodes a proteinase gene. A detailed restriction and deletion map of pLP712 has been determined and the introduction of two deletions of the plasmid was used to generate a mini-lactose plasmid pMG820 that was only 23.7 kb in size [20]. This plasmid retained normal conjugal transfer properties and still generated progeny with cell aggregation and high transfer capability. As with the S. lactis ML3 system, these progeny contained enlarged plasmids, which were analysed by restriction endonuclease digestion. The data obtained revealed a significant difference from the situation in S. lactis ML3 in that the size of the enlarged plasmids varied. The foreign DNA inserts into pMG820 always mapped to within a discrete region but the insert DNA, whilst clearly involving a common sequence, showed a complex variability. Amongst the many different enlarged plasmids examined, groups of more closely related plasmids could be defined. Within such a related group, the insert DNA was clearly the same except that its length varied, but only from one end. One end of the insert DNA was thus fixed, but the other appeared to vary in an apparently random way (Gasson and Maeda, unpublished). This information is compatible with the more complete explanation for cointegrate plasmid formation in S. lactis ML3, if a sex factor equivalent to pRS01 was located in the chromosome of S. lactis 712. Under those circumstances, the lactose plasmid pMG820 would become integrated within the sex factor on the chromosome as a result of transposition associated with an insertion sequence equivalent to ISSIS. A situation similar to that found in an E. coli Hfr strain would exist with DNA transfer potentially beginning from an origin of transfer on the sex factor and resulting in the transfer of the contiguous chromosomal DNA. Selection for lactosepositive progeny would lead to the isolation of recircularised plasmids that would be equivalent to E. Coli F-prime plasmids which might be formed in the donor prior to conjugation. Under such circumstances the related groups of plasmids observed amongst S. lactis 712 progeny would have arisen from one particular type of insertion of pMG820 into the chromosomally located sex factor. Subsequently, a family of related plas-

Gene transfer systems and transposons mids would be generated by separate recircularisation events. The creation of progeny with restriction patterns that do not fit into the same family is simply explained by their creation from a distinct primary insertion of pMG820 into a different site of the chromosomally located sex factor. The enlarged plasmids found in aggregating strains derived from transconjugants of S. lactis ML3 and S. lactis 712 are unstable and they readily revert to a non-aggregating phenotype. Anderson and McKay [56] analysed the molecular events involved in this reversion to a nonaggregating phenotype in S. lactis ML3 progeny. The predominant event appeared to be insertion of the plasmid cointegrate into the chromosome, but precise excision of pRS01 from the lactose plasmid pSK08 and imprecise excision (leading to the creation of intermediately sized novel plasmids) were also observed. Another interesting cause of loss of the aggregation phenotype was an inversion of a DNA sequence within pRS01, which did not affect the size or the copy number of the plasmid cointegrate. Inversion led to a reduction in transfer frequency by a factor of 10-4 as well as loss of clumping. Selection of progeny using such a plasmid as donor caused the reappearance of the aggregation phenotype and this was shown to be due to another inversion event that recreated the original plasmid cointegrate. These observations are further discussed below in the section on transposable elements. The wide host range plasmid pAM//1 has been used widely to demonstrate conjugal transfer between many strains of lactic acid bacteria [30, 42, 58-62]. In some cases, such as transfer into Pediococcus [63], alternative plasmids such as piP501 have proved to be more effective. The frequency of transfer observed can often be rather low and recently van der Lelie and Venema [64] have shown that conjugal transfer of pAMI31 from S. lactis into Bacillus subtilis leads to a specific 10.6 kb deletion of pAM/31 that prevents subsequent conjugal transfer of the plasmid. Plasmid pAMB1 has been used to mobilise other plasmids that encode genes for industrially important phenotypes. For example, Hayes et al. [65] used pAM/31 to transfer genes for proteinase production from S. cremoris strains UC317, UC205 and UC411 into S. lactis. They found that transconjugants contained novel recombinant plasmids, some of which were pAMI31 cointegrates. It has also been observed that pAMB1 will mobilise the lactic

495

streptococcal vector pCK1 at a low frequency in S. lactis (M. Gasson, unpublished). The exploitation of wide host range transmissible plasmids to mobilise gene cloning vectors has been developed in a more sophisticated way. Smith & Clewell [411 first described a means of introducing cloned DNA into Streptococcus faecalis using conjugative mobilisation. This approach involved two partially homologous plasmids, the Escherichia coli-streptococeal vector pVA838 and conjugative plasmid pVA797. A strain of Streptococcus sanguis already carrying pVA797 was first transformed with pVA838 and through homologous recombination a cointegrate was formed which was then transferred into S. [aecalis by conjugation. Subsequently, the cointegrate was resolved and the plasmids segregated due to replicon incompatibility to give rise to transconjugants carrying only pVA838. It was shown that S. faecalis DNA cloned onto pVA838 in E. coli could be returned to S. faecalis via S. sanguis using this technique. Romero et al. [42] extended the use of vector mobilisation to the lactic acid bacteria. A transformable S. lactis strain acted as an intermediate to effect the mobilisation of vectors pVA838 and pSA3 by pVA797 into various strains of S. lactis, S. cremoris, S. lactis ssp. diacetylactis, S. thermophilus and S. faecalis. An improved vector pTG222 was also constructed which h a d u n i a1u e cloninotD giteg for

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endonucleases EcoRI, Smal, Banll, Sacl, Sph 1, Kpn 1, Xba 1 and BamHI and this was used to demonstrate the conjugal mobilisation of a cloned S. thermophilus gene for/3-galactosidase. An interesting example of an industrially significant improvement brought about by conjugal transfer is the selection of 'super-nisin' producing strains. Tsai and Sandine [49] described the transfer of sucrose fermentation and nisin production genes from S. lactis 7962 into strain of Leuconostoc dextranicum. One transconjugant was found to produce excessive amounts of nisin. It was highly resistant to nisin, produced inhibition zones 167% larger than S. lactis 7962 and an assay for nisin activity revealed a 1000fold increase in yield. The use of conjugation to construct improved strains for use by the dairy industry has recently been demonstrated by Sanders et al. [53]. This conventional genetic approach has the advantage of avoiding some of the problems that are associated with acceptance of genetically engineered strains for food use. It was shown by Sing and Klaenhammer [51] and Jarvis and Klaen-

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G. F. Fitzgerald and M. J. Gasson

hammer [66] that plasmid pTR2030, which confers bacteriophage resistance in S. lactis ME2 could be transferred by conjugation into S. cremoris strains. The temperature sensitivity of this bacteriophage resistance mechanism that was found in S. lactis was not expressed in the S. cremoris transconjugants and the plasmid was thus of industrial significance. Sanders et al. [53] adopted a progeny selection approach that used the lactose-positive character of the S. cremoris strain and thus avoided the need to introduce a counter-selective genetic marker. They were thus able to isolate good fast acid-producing progeny strains with the addition of bacteriophageresistance conferred by pTR2030. This is an excellent example of the potential of genetic manipulation for industrial strain improvement in the lactic acid bacteria.

Transposable elements in lactic acid bacteria Transposable elements may be defined as discrete segments of DNA that are capable of moving or transposing from one replicon to another. The first transposable elements discovered and investigated were the controlling elements in maize (Zea mays) identified by McClintock [67, 68]. Subsequently, mobile elements have been identified in Drosophilia, yeasts and almost all bacterial genera [69]. Essentially, two types of transposable element have been identified, i.e., transposons and insertion sequences. Both generally share a'common structure whereby they encode a transposase enzyme and are flanked by inverted repeated sequences, although there are notable exceptions to this latter rule, such as bacteriophage Mu [70]. Transposons also carry an additional gene, usually encoding a readily detectable phenotypic marker, such as antibiotic resistance, and consequently these are larger than insertion sequences [70-73]. Transposons themselves can be divided into a number of classes but in the context of this review we will be concerned with the so-called conjugative transposons which have recently been shown to offer considerable potential as tools for the genetic analysis of the lactic acid bacteria. These conjugative elements appear to be unique to members of the genus Streptococcus; none as yet has been identified in the lactic streptococci. Nevertheless, they have been introduced into these bacteria by conjugation and their abilities to target and mutate

genes, particularly those of chromosomal origin, have been demonstrated [74-78, 93, 94]. However, before discussing the application of these conjugative transposons to the lactic acid bacteria, we will examine the evidence demonstrating the presence of native transposable elements in these bacteria and we will review the excellent work of Shimizu-Kadota and her colleagues, who have discovered the insertion sequence ISL1 in Lactobacillus casei. Recombination and rearrangements of DNA have been observed to occur commonly in the lactic streptococci, particularly after gene transfer has taken place. These are exemplified, for example, by the appearance of novel plasmids following conjugation [15, 34, 35, 43, 79, 80] and transduction [11, 18, 25]. Other observations, such as the stabilisation of L a c / P r t in transductants [17, 22, 25], the failure to detect plasmids in transconjugants following the conjugative transfer of traits which are plasmid coded in the donor [43, 46], and the inherent instability of some lactic streptococcal plasmids [14, 20, 26, 56] can most easily be explained by recombination events within and between DNA molecules. These could be mediated by the host's recombination (Rec) machinery (i.e., homologous recombination) or by illegitimate or transposontype recombination mediated by transposable elements. In the lactic streptococci, these questions have been investigated most rigorously in S. lactis ML3, where Anderson and McKay [26, 56] have examined the formation of large recombinant plasmids following conjugation. Detailed restriction mapping has shown that a novel 104 kb plasmid observed in ML3-derived transconjugants was a cointegrate molecule arising from the fusion of the individual plasmids pSK08 (55 kb, Lac) and pRS01 (48.4 kb, associated with clumping and transfer characteristics) present in the parental ML3 donor. Anderson and McKay [26] explained this observation on the basis of an insertion sequence on pSK08 which, during transposition to pRS01, replicated leaving a copy at each junction of the cointegrate and, as described earlier, this theory has been proven (see section on conjugation). It is interesting that in addition to the IS element on pSK08, an invertible DNA region was observed within pRS01 and the orientation of this sequence was shown to dictate the expression of clumping genes (which promote high frequency conjugation). Although these invertible sequences have not been characterised in detail,

Gene transfer systems and transposons

it is likely that they operate in a manner similar to the invertible G segment of Mu and the invertible H segment of Salmonella typhimurium, which control tail fibre biosynthesis (and consequently adsorption properties of bacteriophage Mu) and flagellar phase variation, respectively [81, 82]. Recombination events analogous to those observed in S. lactis ML3 have also been described in the related S. lactis 712 [13-15, 17]. The restriction data generated for pSK08 and pLP712 (i.e., the Lac plasmids of strains ML3 and 712, respectively) indicate that both plasmids are very similar, if not identical, and Gasson and Davies [17] have shown that DNA insertions occur in a specific 3 kb region of pLP712 which, according to Kondo and McKay [21], corresponds to an equivalent region of pSK08. The origin of the inserting DNA in the 712 system is not proven but, as described earlier, Gasson and Davies [17] have suggested that it is a chromosoreally located sex factor equivalent to pRS01. It is tempting to speculate that transposable elements are common in lactic streptococci. Their widespread occurrence would help explain the commonly observed in vivo rearrangement of DNA in these bacteria. Electron microscope and sequence analysis will be required to confirm the presence of these elements and to determine if their structure is similar to the classical insertion elements of E. coli, for example. Shimizu-Kadota and her colleagues have used these techniques to characterise a new insertion sequence, ISLI, in Lactobacillus casei $1 used in lactic acid fermentations in Japan. When investigating the origin of a virulent phage for the LactobaciUus culture, they were able to show that it was derived from a prophage resident in this host [83]. Detailed comparison of selected virulent phage DNAs with prophage DNA, using restriction analysis, led to the identification of inserted DNA in the prophage genome and, since this was the only difference between the phage types, it was held to be responsible for the conversion of the lysogenic phage to virulence [841. Heteroduplex and sequence analysis confirmet~ that the insertion was a transposable element which they designated ISL1 [85]. The insertion sequence is 1256 bp long, contains at least two open reading frames of 279 and 822 bases on one strand (ORF1 and ORF2, respectively) which have a coding capacity for proteins of 10.7 and 31.7 kDa, respectively. The element was found to have an inverted repeated sequence at its ter-

497

mini and was bracketed by 3 bp direct repeats of the target DNA. Hybridisation analysis showed that ISL1 was derived from the L. casei chromosome. Interestingly, no significant homology with the known insertion sequences of E. coli and Halobacterium was found, nor did the element appear to possess the canonical E. coli promoter or transcriptional terminator sequence. However, a sequence similar to the E. coli ribosomal binding site was found 8 bp upstream from the putative start codon of ORF1. ShimizuKadota et al. [85] speculate that the insertion sequence may be responsible for the acquisition of virulence by the prophage due either to inactivation of operator(s) or to the provision of a new promoter for the phage genome which is functional in the presence of the phage repressor resulting, in either case, in the constitutive expression of genes essential for lytic growth of the lysogenic phage. These studies with L. casei represent a major advance, not just in the demonstration of transposable elements in lactic acid bacteria, but also in the application of the techniques of molecular biology to show that lysogenic phage can be a source of virulent phage in commercial practice. A recent development in genetic studies of lactic acid bacteria has been the application of 'transposon technology' for genetic analysis. The transposons used are members of the Tn916 family of transposable elements whose principal properties are listed on Table I. A number of these properties facilitate the exploitation of these transposons for the targeting and cloning of genes, using a strategy originally proposed by Gawron-Burke and Clewell [86]. This strategy is outlined in Fig. 1. Essentially, it involves the introduction of Tn916 or one of its close relatives into a recipient strain and selecting for trans-

Table I° Properties of Tn916-1ike transposable elements. 1. Isolated from members of the genus Streptococcus 2. Encode tetracycline (terM) resistance 3. 15-16 kb in size 4. Conjugati~ve,.at, frequencies between 10-6-10 -9 per recipient 5. Can be cloned in E. coli which express tetracycline resistance. Absence of selective pressure results in excision and loss of the transposon

G.F. Fitzgerald and M. J. Gasson

498 OONOR

Tn916

RECIPIENT j

GENE X

Screen Tc r transconjugants for mutants

1

Clone EcoRi fragment

in X

in E.col._~i

L

Transposon excises in the absence of Tc

----$tructural integLrity of gene X restorea

Fig. 1. Outline of strategy proposed by Gawron-Burke and Clewell [86] for the targeting and cloning of streptococcal genes using Tn916 or related transposons.

poson encoded tetracycline-resistant (Tc a) transconjugants. These can then be screened for desired mutations which will have arisen due to insertional inactivation. Mutated transconjugant DNA can then be shotgun-cloned into an appropriate vector (i.e., does not have a tet gene), using one of a number of restriction en'donucleases that do not have a recognition sequence within the transposon (e.g., EcoRI, PstI, BamHI, etc.) [87]. Thus, DNA flanking the transposon which contains the interrupted gene of interest will be cloned. Recombinant molecules can be transformed into E. coli and transformants selected on the basis of the transposon encoded Tc resistance. Since growth of Tc r clones in the absence of tetracycline results in the high frequency and precise excision of the element [86], the structural integrity of the gene of interest is restored and the gene is now available in a background to allow easy analysis and characterisation. This gene can then be returned to its natural host under controlled conditions or can be used as a probe to identify similar genes in other hosts. Although this strategy was originally proposed and developed for Tnglr, the related Tn918 [88] and Tn919 [73] can also be used in a similar manner, since they display the same critical properties as Tn916

(i.e., conjugative functions and excision in E. coli in the absence of tetracycline). Tn916 has already been used to generate variants altered in S. faecalis sex pheromone production [89] and in S. pyogenes streptolysin S production [90]. Clewell et al. [91] also used Tn916 to locate haemolysin genes on the S. faecalis plasmid pAD1. Since it is not within the scope of this article to review conjugative transposons per se, as they do not originate in dairy lactic acid bacteria, we direct those interested to the recent excellent review of Tn916 and other conjugative transposons by Clewell and Gawron-Burke [92]. The properties of the Tn916-1ike elements suggest obvious applications for the genetic analysis of Gram-positive bacteria, particularly those in which genetic systems are not yet well developed, such as the lactic acid bacteria. In the laboratory of one of the authors (GFF), we have examined the conditions required for the successful use of Tn919 in the lactic streptococci and we have shown that this element can be employed for the targeting of genes, including those of chromosomal origin. Tn919, which was originally identified in S. sanguis FC1 [74], shares all the properties of Tn916 (Table I) and structurally, both transposons are similar even though there are some differences in their restriction maps [92] (C. Hill, personal communication). The criteria required for the successful use of Tn919 as a genetic tool include a wide recipient host range among the lactic streptococci, reasonable transfer frequency to these recipients to facilitate mutant isolation and random insertion of the element into recipient chromosomes so that any single gene will have an equal probability of being insertionally inactivated. Tn919 has been transferred in agar surface matings into S. lactis, S. cremoris and S. lactis ssp. diacetylactis strains at frequencies ranging between 5.0 x 10-5 and 4.0 x 10 -8 per recipient using an S. faecalis or S. lactis donor [74, 76-78]. Transfer to Lactobacillus plantarum and Leuconostoc mesenteroides has also been reported [76]. In addition, Tn916 has been introduced into S. lactis [75, 93, 94]. However, the frequencies of conjugative transfer of Tn919 observed with lactic streptococcal recipients are generally too low for practical mutant screening purposes. To address this problem, Hill et al. [77] have developed a hig'n fz~.quency delivery system for Tn919 to selected recipient strains. This exploits the high frequency conjugative properties of pMG600, which is a deletion derived of pLP712

Gene transfer systems and transposons

(the L a c / P r t plasmid of S. lactis 712) and which confers the Lac +, Lax- phenotype (Lax- refers to the capacity of a strain to clump in broth [31]). When pMG600 was introduced into S. laciis CH919 (i.e., an S. lactis MG1363 strain containing Tn919 on its chromosome) a selected transconjugant, designated CH001, was capable of transferring Tn919 at a significantly elevated frequency compared to transfer in the absence of pMG600 (1.3 x 10-4 compared to 7.0 x 10-8 per recipient). Furthermore, the presence of pMG600 allowed transfer to occur on agar surfaces (transfer of Tn919 alone occurs only on nitrocellulose filters) and this allowed a greater recovery of recipients from the agar plates. In practice, this strategy resulted in a 1000-10000fold increase in transconjugant numbers per mating experiment. Examination of the transconjugants showed that approximately 80% exhibited the Lac + Lax- Tc R phenotype, suggesting transfer of both Tn919 and pMG600, whereas 20% were Lac- Lax + Tca, suggesting transfer of Tn919 alone. In addition, the high frequency transfer was observed even when pMG600 was present in the recipient rather than the donor strain. These data imply that pMG600 is providing conjugative functions for Tn919 which are otherwise poorly expressed by the transposon. It also suggests that the plasmid and transposon do not form any cointegrate type structure during the transfer event. Similar high frequency transfer of TriO10 into S. lactis ssp. diacetylactis 18-16 has been observed, but S. lactis CH001 was unable to transfer the transposon into S. cremoris, Lactobacillus or Leuconostoc strains on agar surfaces. This indicated that the high frequency delivery system is somewhat strain specific and that the conjugal functions specified by pMG600 are incompatible with some recipient cell types. A further and essential requirement for the application of Tn919 in targeting lactic streptococcal genes is that insertion into the recipient chromosome be random. The nature of Tn919 insertion in a number of strains has been investi: gated by comparing the sizes of HindIIIgenerated chromosome-transpo:~on DNA junction fragments (after transfer of the fractionated DNA to nitrocellulose filters) using a labelled Tn919 probe. Since Tn919 itself has only a single HindIII site, each insertion will result in two junction fragments tliat will light up after probing and the size of these fragments is an indication of the insertion specificity, i.e., similarly sized fragments in all transconjugants indicate

499

site-specific insertion. Examination of a limited number of strains suggests that the nature of Tn919 insertion is host determined. While insertion into S. lactis SK3, S. cremoris UC317 (previously named S. cremoris 17) and S. lactis ssp. diacetylactis 18-16 was random, occurring in chromosomal and plasmid DNA [77, 78], sitespecific insertion was observed in the chromosome of all transconjugants derived from S. lactis MG1363 (a plasmid-free derivative of S. lactis 712) examined thus far, which suggests that the chromosome of this strain contains an insertion hot-spot for Tn919 [76]. Site-specific insertion was also demonstrated by Tn916 when transferred from S. faecalis into S. mutans 6715 [87], so this phenomenon is not unique to S. lactis MG1363. However, the basis for the specificity in these systems is not known. Hill et al. (unpublished data) have used Tn919 to conduct insertional inactivation experiments on chromosomally located genes in S. lactis ssp. diacetylactis 18-16, an ideal subject strain for analysis, since insertion is random in its chromosome and it is compatible with the pMG600aided high frequency transfer system. Initial studies have concentrated on a eitritase (EC 4.1.3.6.) deficient mutant which has a single copy of Tn919 in its chromosome. This enzyme converts citric acid into acetic and oxaloacetic acids in the citrate fermentation pathway. DNA flanking the transposon has been cloned in L. coli, using a modification of th~ ~awran-l:lurka and Clewell [86] cloning strategy described earlier. The related transposon Tn916 has beenused successfully to identify genes involved in the maiolactate, maltose, mannose and arginine metabolic pathways in S. lactis IL1441 [93] (P. Renault, personal communication). Insertion of Tn916 into the IL1441 chromosome was random and frequently occurred at multiple sites. In the case of the malolactate-defective isolates, three classes of mutants were isolated at a frequency of 10-3-10 -4, corresponding to mutations in structural, transport and regulatory genes. When Tn916 mutated DNA from representative mutants was cloned in E. coli, rearrangements and deletions of the cloned DNA were observed in many instances. Nevertheless, DNA from two clones was stable in the E. coli host and in the case of one of these the insertio~ally inactivated fragment could be regenerated by spontaneous (and probably precise) excision of the transposon (P. Renault, personal communication). Loureiro dos Santos and Cho-

500

G . F . Fitzgerald and M. J. Gasson

pin [94] have also demonstrated the feasibility of using the transposon-based cloning strategy in lactic streptococci. Using the plasmid vector pIL204 (a deletion derivative of pAM/]I), a 22 kb chromosomal fragment from S. lactis, containing Tn916 and host DNA, was cloned directly into an S. lactis IL 1403 recipient by protoplast transformation. It has also been established that Tn916 inserts at various sites in the chromosome of Tc R IL 1401-derived transconjugants and in a number of instances double insertions of the element were observed (A. Chopin, personal communication). The Tn916 family of transposons offers exciting possibilities for the genetic analysis of the lactic acid bacteria. Their ability to target chromosomal genes in the lactic streptococci is particularly encouraging, since these have previously received relatively little attention. However, the site-specific insertion phenomenon observed in some strains may limit their application somewhat, but ensuring that insertion is random in the strain(s) under investigation will safeguard against this possibility. In addition, the large size of these transposons (15-17 kb) may lead to difficulties when cloning the elements plus flanking host DNA, but these can be alleviated by judicious choices of vectors, restriction endonucleases and recipient strains. Recently, Courvalin and Carlier [95] have described a 25.3 kb transposon, Tn1545 from S. pneumoniae, which encodes multiple drug resistance and which is capable of promoting its own transfer into a wide range of hosts including S. lactis, S. cremoris and S. lactis ssp. diacetylactis. This transposon may have similar applications in these bacteria to the Tn919-1ike elements, although its larger size may restrict its use for cloning. Conjugative transfer of antibiotic resistance determinants in the absence of plasmid DNA is common in the genus Streptococcus (see Clewell and Gawron-Burke for an overview of this topic [92]). Although the involvement of transposons has been positively established in only some instances, it is likely that this type of gene transfer is generally transposon mediated. The failure to detect such elements in lactic streptococci may reflect the fact that, for evolutionary reasons, transposons are specifically associated with drug resistance determinants which are not encountered in the dairy streptococci (excepting nisin, which is not used in human or animal medicine). Furthermore, the conjugative elements observed in other species of streptococci usually reside

on the host chromosome and, since the emphasis of genetic studies in the lactic streptococci has been on plasmid-located genes, it may be that such elements, if present, still await discovery.

Concluding remarks The availability of in vivo gene transfer systems has been of significant value in the advancement of genetic studies of the lactic acid bacteria, particularly the lactic streptococci. While transduction and conjugation have been used very successfully as tools to examine the role of plasmids in these bacteria, many fundamental questions relating to the transfer systems themselves have not been addressed. Conjugation appears to be an extremely complex and interesting phenomenon but is as yet poorly understood and will surely be the focus of greater research interest in the future. The application of conjugation to study plasmids in lactic streptococci has also highlighted the fact that genetic material in these bacteria is undergoing constant rearrangement and structural alterations. The recent application of more sophisticated analytical techniques is beginning to provide answers to explain the molecular basis for some of these events and it is interesting, but perhaps not surprising, that transposable elements appear to play a key role in mediating these rearrangements. Conjugation, a natural and widely occurring gene transfer system, also appears to be an attractive mechanism for generating genetically modified and improved strains for industrial use without having to resort to the less readily accepted recombinant DNA technologies. In this regard, the development of phage-resistant starter strains, following the introduction of plasmids mediating the resistance, is a major achievement [53]. A similar approach used to construct a super-nisinproducing strain of Leuconostoc may also have practical significance in food fermentation [49]. The application of conjugative transposons of the Tn916 family is an exciting development in the genetic analysis of the lactic acid bacteria. The demonstration of a wide recipient host range, random insertion into the genomes of most strains examined and, in particular, an r~oility to insertionally inactivate chromosomal genes suggests that these elements may be of use in unlocking what has been until recently a 'black box' - i.e., the chromosome of lactic acid

Gene transfer systems and transposons bacteria. W h e t h e r they fulfil their promise of being useful in the cloning of both chromosomal and plasmid genes remains to be seen.

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/1~¢;~...~

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