Actttatgaatataaagtatagtgtgttatactttacatg

F-like (Jaworski and

Clewell, 1995) IncP (An and Clewell,

1997)

pMV158 (Josson et al., 1990)

1998)

IncP (Van Kranenburg and De Vos, 1998)

IncQ (Wang and Mac-

rina, 1995) pMV158 (Guzman and Espinosa, 1997)

Inverted repeat sequences and defined nic sites are indicated by horizontal and vertical arrows, respectively. Abbreviations: c = conjugative (self-transmissible), m = mobilizable.

Classification scheme based on nucleotide sequence homology to oriT regions from gram-negative F-like, IncP, or IncQ plasmids, or from the streptococcal plasmid pMV158.

The Tn916 oriT has been localized to a 466-bp fragment, but the sequence displayed is actually one of three sites in this region that show homology to the nic regions of F-like (shown) or IncP plasmids.

Identification of this oriT region is based entirely on sequence homology to pMV158 (Guzman and Espinosa, 1997).

The pRS01 oriT has been localized to a 446 bp Pstl-Xbal fragment, but the displayed sequence is actually one of five sites within this region that show homology to the nic regions of IncQ (shown), F-like, or IncP plasmids. pNZ4000 contains two identical and functional copies of this sequence.

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c c m c m i c i m d g to recognize that many conjugation genes from LAB lack significant homology to any known proteins. Although these observations may largely reflect the mechanistic differences that are imposed by absence of pili, it is also plausible that some processes for DNA transfer and mating pair resolution in gram+ bacteria are quite different from those seen in gram" cells and even from one another (Dougherty et al, 1998; Wang and Macrina, 1995). For this reason, it is encouraging to note recent growth in nucleotide sequence data for conjugal elements in dairy LAB (Burrus et al., 2000; Dougherty et al., 1998; Godon et al., 1994; Mills et al., 1996, 1998; Van Kranenburg and De Vos, 1998), because this information should stimulate more fundamental examinations of conjugation in these very important bacteria.

D. Transformation

Transformation is the process wherein free DNA molecules are introduced into cells. The power of an efficient and reproducible transformation system is that it permits us to manipulate genes in vitro and then analyze the consequences on in vivo molecular and cellular functions. Many bacteria, including some species of nondairy streptococci, can assume a ''competent'' state that allows them to take up DNA from their environment (Havarstein et al., 1997). This ability is determined by a set of unique genes that encode proteins for extracellular DNA binding, uptake, and integration. Expression of host competence genes is induced when the concentration of a host-secreted, competence-stimulating peptide (i.e., a competence pheromone) in the medium reaches a critical threshold. Natural competence has not been demonstrated in any of the food-grade LAB, but Bolotin et al. (1999) recently reported that the Lc. lactis genome appears to contain a complete set of competence genes.

In the absence of natural competence, the most effective method for transformation in most bacteria is electroporation. When cellular membranes are exposed to a high-voltage electric field, they become polarized and a voltage potential develops across the membrane. Electroporation technology is based upon the discovery that when this potential exceeds a certain threshold, localized breakdown of the membrane forms pores that render the cell permeable to extraneous molecules (Ho and Mittal, 1996). Under conditions that may be established experimentally, pore formation is reversible and cells remain viable. The mechanism for entry of DNA or other molecules into cells by electroporation is still unknown, but the availability of inexpensive and reliable commercial equipment has made electroporation the method of choice for transformation of many bacteria, fungi, and higher cells (Lurquin, 1997).

The first reports of transformation by electroporation (electrotransforma-tion) in dairy LAB appeared in 1987, and by the end of that decade the technology had been successfully applied to Lc. lactis, S. thermophilus, and many species of Lactobacillus and Leuconostoc (Chassy and Flickinger, 1987; David et al., 1989; Harlander, 1987; Hashiba et al., 1990; Luchansky et al., 1988; Powell et al., 1988; Somkuti and Steinberg, 1988). One of the most encouraging observations to emerge from this and subsequent research is that a single electroporation protocol can often effect transformation of different strains and even different genera of LAB. Thus, even though parameters for optimal electrotransformation of an individual strain will usually need to be established, a general protocol can frequently provide the starting point for such research.

Another important finding is that electrotransformation frequencies are frequently higher and more reproducible if the thick murein layer is weakened before electroporation (Bhowmik and Steele, 1993; Buckley et al., 1999; Dunny et al., 1991; Hashiba et al., 1990; Holo and Nes, 1989; Posno et al., 1991; Powell et al., 1988; Walker et al., 1996; Wei et al., 1995). This is usually achieved by propagating cells in a medium that contains relatively high concentrations of glycine or D/L-threonine, which interfere with cell wall synthesis and assembly. It should be recognized, however, that inhibition of cell wall synthesis is not essential for efficient electroporation of some LAB, and in certain instances it may even be counterproductive (Berthier et al., 1996; Luchansky et al., 1988; Marciset and Mollet, 1994; Wycoff et al., 1991).

Today, representative strains from virtually all industrially important dairy LAB species have been successfully transformed by electroporation, but individual strains from some species—and particularly lactobacilli—are still difficult or even impossible to transform by any known method. Moreover, even among LAB that can be electroporated, only a very few strains can be reproducibly transformed at frequencies greater than 104 transformants per microgram of exogenous DNA (Berthier et al., 1996; Holo and Nes, 1989; Marciset and Mollet, 1994; Posno et al., 1991; Wycoff et al., 1991). Some factors that appear to limit efficiency of electrotransformation in LAB include (1) culture growth phase, concentration, and membrane lipid composition; (2) host-encoded restriction/modification systems; and (3) vector size, purity, and compatibility with endogenous host plasmids (Aukrust and Blom, 1992; Hashiba et al., 1990; Luchansky et al., 1988; Posno et al., 1991; Van der Lelie et al., 1988). Regardless of its molecular basis, the broad variability in electroctransformation efficiency that exists among dairy LAB is unfortunate, because the proficiency at which cells can be transformed is directly related to the ease and flexibility by which recombinant DNA technologies can be employed for genetics research. It is largely for this reason that many LAB researchers pursue a strategy wherein gene cloning and characterization are done in Es. coli, where electrotransformation efficiencies commonly exceed 108/|J.g DNA, after which time DNA constructs are moved into the LAB of interest by electroporation. This approach suffers from several limitations, however, and genetics research in dairy LAB would clearly profit from a more fundamental understanding of electrotransformation in these species.

1. Gene Delivery Systems

Vectors for gene cloning in dairy LAB can be divided into two fundamental categories: (1) extrachromosomal vectors that maintain cloned DNA on an autonomously replicating plasmid and (2) integrative vectors that are designed to insert cloned DNA into the host chromosome. The definitive differences between these elements are that the latter group are incapable of independent replication in the host species of interest (i.e., suicide vectors), and they contain specific sequences that promote vector integration into the host chromosome (see below). Some features common to both types of cloning vectors include (1) they encode a selectable phenotype that allows transformed cells to be easily distinguished from nontransformed cells; (2) they possess a ''multiple cloning region'' that is rich in unique restriction endonuclease cleavage sites and where foreign DNA can be inserted into the vector without damage to replication/integration or selection functions; and (3) they are usually small so that recombinant constructs can be more easily transformed into host cells. Some cloning vectors will also encode a second selective phenotype that is abolished by DNA insertions in the multiple cloning region. Loss of that phenotype is then used to discern transformants that contain recombinant molecules from those that only acquire vector DNA.

a. Replicative Vectors The first cloning experiments in dairy LAB employed replicative vectors that were developed for nondairy streptococci and en-terococci, but a number of high- and low-copy number replicative vectors have since been built from the RCR and theta plasmid replicons found in dairy species (De Vos and Simons, 1994; Kondo and McKay, 1985; Von Wright and Sibakov, 1998; Wang and Lee, 1997). Many of these vectors (particularly those based on RCR replicons) (see Sec. II.A.1) have a broad host range and therefore offer the added advantage of serving as shuttle vectors for B. subtilis or Es. coli, where DNA manipulation techniques are particularly well established.

In addition to simple replicative vectors, identification and characterization of LAB gene expression signals and regulatory sequences has permitted construction of more specialized cloning vectors designed to facilitate constitutive or inducible expression of foreign DNA or heterologous protein secretion (De Vos and Simons, 1994; Kahala and Palva, 1999; Kok, 1996; Savijoki et al., 1997; Venema et al., 1999). Access to effective gene expression and protein secretion systems for dairy LAB is a particularly important advancement, because one of the most economically significant applications of biotechnology involves use of microorganisms to produce large amounts of industrially useful proteins. The worldwide industrial enzyme market, for example, has a value in excess of

$1.2 billion per year (excluding pharmaceutical uses) with food industry applications comprising 40% of this market (Williams, 1998). Most of these enzymes are produced by fermentation with genetically modified bacteria, yeasts, and molds, and it is reasonable to assert that food-grade microorganisms such as dairy LAB may offer unique advantages as unicellular factories for production of enzymes (or other proteins) that are intended for use in human food.

b. Integrative Gene Cloning As is outlined in Section II.A.1, native plas-mids and replicative vectors are vulnerable to segregational and structural stability problems that can result in permanent loss of plasmid-coded traits. Integration vectors avoid this problem by recombining with the host chromosome. These constructs are typically assembled in a permissible host such as Es. coli, and then transferred by electroporation into the (nonpermissive) LAB of interest. Two mechanisms that have been used to direct random or site-specific vector integration into the LAB chromosome include IS-mediated transposition and the int-attP functions from temperate bacteriophages, respectively (see Sec. II.B and II.D.2 for details and references). The most common scheme for vector integration in dairy LAB, however, relies on host mechanisms for homologous DNA recombination (Leenhouts, 1990). These systems typically contain a fragment of the LAB host chromosome which serves as a substrate for site-specific, homologous DNA recombination via single- or double-strand crossover. Single crossover recombination results in integration of the entire vector, whose sequence will be flanked by direct repeats of the cloned chromosomal fragment. One of the consequences of single crossover plasmid integration is that the homologous repeats formed by integration make the entire structure susceptible to gene amplification.

In contrast, double crossover recombination results in the exclusive integration of vector sequences that lie between the two recombination sites, with concomitant loss of the corresponding region of the native host chromosome and any extraneous vector sequences. Thus, double crossover recombination is often called replacement recombination. Unfortunately, replacement recombination is a low-frequency event, which limits its application in strains that suffer from a poor transformation efficiency. To overcome this problem, many researchers have abandoned suicide replicons in favor of vectors that display conditional (e.g., temperature-sensitive) replication in the LAB host of interest (Bhowmik and Steele, 1993; Low et al., 1998; Maguin et al., 1992). With these molecules, transformation efficiency and integration events can be uncoupled as transformants are selected under conditions that permit autonomous replication. Next, single crossover integrants are obtained by shifting a population of transformants to nonpermissive conditions, and then a second crossover event is stimulated by returning integrants to the permissive environment.

Aside from their applications in DNA cloning, integration vectors—partic ularly those that effect replacement recombination—are also invaluable to functional genetics research. This is because they facilitate the construction, by gene knockouts, of isogenic mutants that differ only by the action of a single polypeptide. By comparing the wild-type culture to its isogenic derivative, the role of that polypeptide (and its gene) in LAB cellular or industrial processes can be unequivocally established.

c. Food-Grade Gene-Cloning Systems More than two decades of intensive and worldwide research efforts have given us a tremendous understanding of biochemistry and genetics in dairy LAB. Important biochemical pathways have been elucidated, gene transfer systems have been developed for many strains, a great number of important genes (even entire chromosomes!) have been characterized at the nucleotide sequence level, and mechanisms for gene expression and protein secretion have been identified. To apply this knowledge toward industrial strain improvements, however, it is imperative that we have gene-delivery systems that of themselves do not present a safety concern in human food applications. The most important attributes of these systems, which are termed food-grade vectors, is that they be genetically well defined and not impart any antibiotic resistance gene to the host bacterium. The latter requirement is readily met by vectors that effect replacement recombination, but integrative or replicative gene-delivery systems whose selectable marker will be retained in the host must encode a food-grade alternative to antibiotic resistance. Examples of food-grade selection systems that have been used to satisfy this requirement include auxotrophic complementation, resistance to nisin or other LAB bacteriocins, and ability to ferment new carbohydrates (Allison and Klaenhammer, 1996; De Vos and Simons, 1994; Hashiba et al., 1992; Leenhouts et al., 1998; Lin et al., 1996; S0renson et al., 2000).

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