Because of its singular economic importance as the starter bacterium for industrial production of Cheddar and Gouda cheeses, and the relative ease by which it can be handled in the laboratory, much of our current understanding of genetics in dairy LAB has come from study of Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris (henceforth jointly described as Lc. lactis). A third subspecies, Lc. lactis subsp. hordniae, is not used as a dairy starter and will not be considered further. In this section, we will examine four types of genetic elements that have been characterized at the nucleotide sequence level in Lc. lactis and, to a lesser extent, other dairy LAB. They include plasmid DNA, transposable elements, bacterio-phages and, most impressively, the bacterial chromosome.
Plasmids are extrachromosomal, autonomously replicating DNA molecules that exist independently of the bacterial chromosome. Molecular and genetic studies of bacterial plasmids have yielded extraordinary insight into cellular mechanisms for DNA replication, gene transfer, gene expression, and genetic recombination. Plasmids have also played an integral role in development and evolution of recombinant DNA technologies for many organisms, including dairy LAB.
Most plasmids are covalently closed circular molecules, but linear plasmids have been reported in several eubacteria including one species of LAB, Lb. gas-seri (Davidson et al., 1996; Meinhardt et al., 1997). The number of copies at which a particular plasmid species exists within a bacterium (i.e., its copy number) varies widely and can range from as few as one or two to tens or even hundreds of molecules (Actis et al., 1999; Clewell, 1981). Under most conditions, plasmid-coded functions are not essential to host survival (exceptions involve properties such as antibiotic resistance that confer a selective advantage under specific environmental conditions), but they may allow the cell to compete better with other microorganisms that share their ecological niche. Therefore, if a daughter cell loses a particular plasmid species through plasmid replication or segregation errors, it will usually continue to grow and may even predominate over its wild-type population. Loss of the plasmid will, however, result in permanent loss of any trait encoded by that plasmid.
The first reports of plasmid DNA in LAB were published in the early 1970s by researchers working with En. faecalis and S. mutans (Clewell, 1981). Among food-grade LAB, it was the long-standing observation that many Lc. lactis dairy starters permanently lost their acid- or flavor-producing phenotypes (and the fact that the frequency of these events was increased under plasmid curing conditions) that served to stimulate the first inquiries into the plasmid biology of these organisms (McKay, 1983). We now recognize that lactococci are an especially fertile source of plasmid DNA, and that genes for many of this bacterium's industrially important traits are encoded by plasmids. The latter discovery enlivened worldwide interest in LAB plasmid biology and genetics, and we now know that plasmid DNA is a frequent component of the genome in leuconostocs, oenococci, pediococci, and some lactobacilli. Plasmids have also been identified less frequently in other food-grade LAB, including Carnobacterium, S. ther-mophilus, Tetragenococcus, and Weissella (Benachour et al., 1997; Brito and Paveia, 1999; Davidson et al., 1996; Martin et al., 1999). The rich diversity of plasmid species in LAB is fortuitous, because it provides a ready source of extrachromosomal replicons to support development of gene-cloning vectors (De Vos and Simons, 1994; von Wright and Sibakov, 1998; Wang and Lee, 1997). In addition, although most of these plasmids are cryptic, several interesting and useful phenotypic properties have been linked to plasmid DNA in food-grade LAB (Table 1).
The segregational and structural stability of extrachromosomal DNA can be influenced by the mode of plasmid replication (Biet et al., 1999; Gruss and Ehrlich, 1989; Kiewiet et al., 1993; Lee et al., 1998), and the industrial significance of plasmid DNA in LAB warrants attention to the molecular biology of plasmid replication and segregation in these bacteria. Characterization of the nucleotide sequence and genetic organization of plasmid replicons in eubacteria has identified five distinct systems for plasmid replication; circular plasmids may replicate via rolling-circle replication (RCR), theta replication, or strand displacement, whereas linear plasmids are thought to replicate through virus-like processes that involve formation of circular intermediates (hairpin plasmids) or protein priming (plasmids with 5'-linked proteins) (Actis et al., 1999; Del Solar et al., 1998; Meinhardt et al., 1997). The replication system(s) employed by linear plasmids of Lb. gasseri have yet to be characterized, but nucleotide sequence and structural analysis of replicons from several circular LAB plasmids has confirmed that these molecules replicate by RCR or theta mechanisms (De Vos and Simons, 1994; von Wright and Sibakov, 1998; Wang and Lee, 1997).
a. Rolling-Circle Replication The most common type of replication system in plasmids from LAB and other gram+ bacteria is RCR, a process that involves synthesis of single-stranded DNA (ssDNA) intermediates (Fig. 1). Because ssDNA is a reactive intermediate in all DNA recombination processes, RCR plasmids are particularly vulnerable to segregational and structural instability (Gruss and Ehrlich, 1989; Kiewiet et al., 1993). As might be expected, this attribute can be problematic to gene-cloning strategies with vectors constructed from RCR plasmid replicons (Biet et al., 1999; De Vos and Simons, 1994; Lee et al., 1998).
Plasmids that replicate by the RCR model have been identified in Lc. lactis, O. oeni, and in several species of lactobacilli, leuconostocs, and streptococci (including S. thermophilus) (Biet et al., 1999; Khan, 1997). These plasmids are relatively small (most are 1.3- 10.0-kb pairs), broad host range molecules (many RCR plasmids from LAB can replicate in Escherichia [Es.] coli) that share several structural features (Khan, 1997). These include (1) a rep gene, encoding an origin-specific replication initiation protein (Rep) that has nicking and religating activities; (2) a double-strand (plus) origin, ori, where Rep nicks the leading strand of DNA to initiate replication and where, after each replicative cycle, Rep nicks a second time to release the leading strand; and (3) a single-strand (minus) origin, sso, where replication of the lagging strand is initiated (and whose recognition appears critical in determining plasmid host range and stability). In addition, RCR plasmids typically encode functions that regulate plasmid copy number. The three most common mechanisms involve the synthesis of a rep repressor protein or production of antisense RNAs that either attenuate rep transcription or inhibit Rep mRNA translation (Khan, 1997).
Amino acid and nucleotide sequence alignments of Rep proteins and their double-strand origins, respectively, have shown that RCR plasmids can be subdivided into at least five families represented by plasmids pT181, pE194, pC194, pSN2, and pIJ101 (Khan, 1997). Thus far, most RCR plasmids that have been characterized in LAB fall within the pE194 and pC194 families, but a few members of the pT181 family have also been identified (Alegre et al., 1999; Biet et al., 1999; Khan, 1997). In addition, several RCR plasmids from LAB and other gram+ bacteria do not belong to any of the five existing families, which suggests that the number of RCR plasmid families will expand as more replicons are characterized (Khan, 1997; Wang and Lee, 1997).
Further research to classify RCR plasmids from LAB will serve to clarify the basic understanding of RCR replicons in general, and it will also benefit applied dairy science because this property can influence plasmid incompatibility (and thus the segregational stability of extrachromosomal gene cloning vectors in LAB hosts with native plasmid DNA). Incompatibility is a term that refers to the inability of independent replicons to coexist stably within the same host cell in the absence of any selective pressure. Plasmids that possess identical replication ro 09
Table 1 Plasmid-Encoded Properties in Food-Grade Lactic Acid Bacteria
Bacteriocin production/immunity Class I: lantibiotics Class II: small heat-stable proteins
Class IV: complex bacteriocins Bacteriophage defense Abortive infection Phage adsorption Restriction/modification Carbohydrate transport/hydrolysis Galactose phosphotransferase (PTS)
Lactose (non-PTS) Maltose PTS Melibiose
Wang and Lee, 1997a) Lb. plantarum, Ln. lactis (De Vos and Vaughan, 1994a; Mayo et al., 1994) Lactobacillus sp. (Chou, 1992) P. pentosaceus (Ray, 1995a) Lb. helveticus (Arihara and Luchansky, 1995a) P. pentosaceus (Ray, 1995a) Lactobacillus sp. (Wang and Lee, 1997a) P. acidilactici, P. pentosaceus (Ray, 1995a)
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