Figure 10.1 Molecular organization of two bacteriophage defense systems, from the native L. lactis plasmid pKR223, that are both dependent on a methylase gene for stabilization in L. lactis. The methylase protects L. lactis from LlaKR2I restriction and also from the abortive infection defense system, AbiR.

10.3.2 Role of Plasmids in Other Dairy Starter Cultures

While plasmids play a lesser role in other dairy starter cultures than in L. lactis, they do confer a variety of phenotypes in many genera. Many isolates do not contain plasmids and others just contain cryptic plasmids. Plasmids have been detected in some dairy Leuconostoc species (38,39). While many Leuconostoc do not contain plasmids, and those that do contain mainly cryptic plasmids, some have been shown to encode metabolic phenotypes. Metabolism of lactose via ^-galactosidase was linked to a plasmid in L. mesenteroides (40) and uptake and metabolism of citrate can also be linked to plasmids (41,42). Recently, diace-tyl (acetoin) reductase was located on a plasmid in a dairy L. pseudomesenteroides starter culture commonly used for the production cultured buttermilk, sour cream, and ripened cream butter (43).

Dairy enterococci starter cultures are somewhat controversial as many enterococci are pathogenic, including members of the two species relevant to the dairy industry, E. faecalis and E. faecium (44). However, isolates used by the diary industry are carefully screened for virulence factors. Nevertheless, studies have shown that nonvirulent strains can acquire virulence factors from other enterococci via plasmid transfer (45,46). Indeed, conjugal transfer of tetracycline and vancomycin resistance genes from enterococci to starter E. faecalis strains during cheese and sausage fermentations has been demonstrated (47). This illustrates that dairy enterococci have to be carefully monitored for safety purposes. While virulent strains of E. faecalis and E. faecium can be problematic, members of both these species are prominent in the human large intestine and are considered my many to have probiotic attributes (48). Plasmids are commonly found in both E. faecalis and E. faecium and while metabolic phenotypes have not been associated with them, they frequently encode bacteriocins (49-53).

Streptococcus thermophilus is the only member of the Streptococcus genus used in dairy fermentations. It is a thermophilic starter culture and historically was primarily a yogurt starter culture, but it is increasingly important as a cheese starter. Plasmids are rare in S. thermophilus, but are found in some strains and in many cases are cryptic. However, a couple of phenotypes have been associated with S. thermophilus plasmids. Some strains have been found to harbor plasmid genes encoding small heat shock proteins (54,55). It is likely that prolonged fermentation pressure at high temperatures provided the selective pressure for acquiring these plasmid genes encoding heat shock proteins via horizontal gene transfer, or reestablishing them on plasmids as a means of increasing expression of this phenotype that is essential for optimum prolonged existence in high temperature environments. Others have been found to harbor R/M systems encoded on plasmids (56,57). Interestingly, R/M systems are the only phage defense systems found in S. thermophilus so far, although possible evidence for an Abi system was suggested previously (58). Heterologous expression of the lactococcal Abi system AbiA has been achieved, but expression could only occur at 30°C and not at the normal growth temperatures of this thermophilic starter (59). It is intriguing that apparently fewer phage defense systems have evolved in S. thermophilus, compared to L. lactis. One possibility is that many phage defense systems either do not work, or function poorly, at elevated temperatures, as illustrated in the AbiA example (59). This appears to be an evolutionary response to prolonged existence in a stressed environment, where there is a need for rapid evolution to exist more effectively in the stressed environment. Eliminating barriers to DNA uptake is an effective means for speeding up evolution in a species. The finding of plasmid encoded hsp genes in S. thermophilus, and not in L. lactis even though its plasmids have been extensively analyzed over the last 30 years, supports this hypothesis.

The genus Lactobacillus covers an extremely broad range of bacteria and the use of lacto-bacilli in the dairy industry is also very broad as they are used in nearly all applications, from yogurt production, cheese production, fermented milks, and as probiotics. They are also the most extensively found bacterial genus in other food fermentations, such as vegetable fermentations (pickles, sauerkraut, and fermented olives), meat fermentations (summer sausage, salami, and pepperoni), bread fermentations (sourdough), wine malolactic fermentations (with another LAB, Oenococcus), and in many fermented foods indigenous to various countries in the world. While plasmids are frequently found in lactobacilli, they are less common in most dairy lactobacilli. The first evidence for plasmids in Lactobacillus was from L. casei in 1976 (60). As Lactobacillus is a very widely studied genus, numerous plasmids have been characterized and numerous phenotypes have been found. Some plas-mids have been linked to the metabolism of sugars, including lactose in some strains of L. acidophilus and L. casei, galactose in L. acidophilus and maltose (reviewed, 61). Citrate utilization has been linked to a plasmid in the plant associated L. plantarum (62). Unlike lactococci, phage defense plasmids are not common in lactobacilli. However, an R/M plasmid was found in a L. helveticus strain (63), and possible evidence for adsorption resistance and Abi plasmids were reported from a nondairy L. plantarum isolate (64). Plasmids have also been linked to exopolysaccharide production in some L. casei strains (65,66). A plasmid harboring resistance to hops, the broad spectrum antimicrobial plant additive, prominent in beer especially India Pale Ale type beers, was found in L. brevis

(67). This is a common beer spoilage organism, and the selective pressure for acquiring hop resistance plasmids was likely due to the presence of hops in its environment. Direct evidence for this, was provided recently by Suzuki et al., who demonstrated that L. brevis strains readily lost their hop resistant abilities when cultured in a hop free environment

(68). Antibiotic resistance plasmids have also been found in lactobacilli, including chlor-amphenicol resistance in L. reuteri (69,70) and L. acidophilus (69), erythromycin resistance in L. reuteri (71,72) and L. fermentum (73), dalfopristin resistance in L. fermentum (73), and tetracycline resistance in L. fermentum (74) and L. plantarum (75). The selective pressure for the acquisition of these antibiotic resistance genes clearly arose from the presence of these antibiotics in their environments. Most of these species are residents of human and animal intestines and the use of antibiotics in humans and animals evidently provided sufficient selective pressure for their acquisition. The one exception is L. plantarum, which is not commonly found in human and animal intestines, but is associated with plants.

However, tetracycline is also used in plant agriculture to prevent bacterial infections in plants (76), thus providing the necessary selective pressure for its acquisition by plant associated microbes. Further evidence is provided by the acquisition of tetracycline resistance by other plant associated microbes, such as Agrobacterium tumefaciens (77). Numerous plasmids have been associated with the production of bacteriocins including, acidocin B (78), acidocin 8912 (79) and acidocin A (80) from L. acidophilus; brevicin 27 from L. brevis (81), Curvacin A from L. curvatus (82), plantacin 154 from L. plantarum (83), and lacticin F from L. johnsonii (84,85). The prominence of bacteriocins in Lactobacillus suggests that this is a major characteristic utilized by this genus for competing against other bacteria in their natural environment. The ability to attain dominance in a natural habitat is directly related to the ability to outcompete other bacteria. It is noteworthy that microbes that are dominant in neutral pH environments do not rely on bacte-riocins to a large extent for competing against other microbes. An interesting example is Bifidobacterium, which is a dairy culture of growing importance because of its probiotic attributes. Bifidobacteria are dominant inhabitants of the large intestine, which is largely a neutral pH environment. However, despite extensive searches by various groups throughout the world, only a few strains of B. bifidum were found to produce a bacteriocin (86,87,88). This illustrates that this genus uses alternative means to compete against other microbes in the large intestine. A likely reason for this is that dominant microbes in neutral pH environments rely on suppressing their competitors by scavenging for the limiting supply of iron available in neutral pH environments (89). This occurs because iron is extremely insoluble at neutral pH and therefore becomes a limiting, essential growth factor (90). Recently it was demonstrated that dominant bifidobacteria in the human large intestine inhibit the growth of their competitors in neutral pH environments, by secreting potent iron binding compounds that scavenge the limiting supplies of iron thus preventing the growth of their competitors (14,91). This effective means for inhibiting the growth of competitors in their natural habitat is consistent with the absence of bacteriocin production by most bifidobacteria. Lactobacilli on the other hand reside in habitats that are acidic, and in those environments iron is readily soluble and therefore not a limiting factor for growth. It is therefore not surprising that most lactobacilli have evolved the ability to produce bacterio-cins to facilitate their dominance in their environment.

The non-LAB dairy cultures, propionibacteria, brevibacteria, and bifidobacteria have not been researched as extensively as the LAB. In all three groups, plasmids are rare, but have been found in some members. The propionibacteria relevant to the dairy industry include P. jensenii, P. acidipropionici, P. freudenreichii (subsp freudenreichii and shermanii), and P. theonii. They are important cultures in many cheese fermentations, especially Swiss cheese. They are also of increasing importance as possible probiotic cultures, because of their ability to survive gastric transport and produce propionic acid, 6-galactosidase, bacteriocins, and vitamin B12, as well as attach to intestinal cells (92,93,94). Plasmids have been reported in some strains of P. jensenii, P. acidipropionici, and P. freudenreichii, and are more commonly found in the latter species. While little research has been conducted on plasmid biology in propionibacteria, most plasmids appear to be small and cryptic. Some do contain useful phenotypes, such as encoding bacteriocin production (95,96).

Brevibacteria belong to the coryneform group of bacteria and are important in the dairy industry particularly for surfaced ripened cheeses, such as Limburger, Brick, Muenster, and some blue cheeses. In contrast to all the other dairy cultures, these bacteria only grow in the presence of oxygen and therefore are only found on the surface of cheeses. B. linens is the primary species of importance to the dairy industry and is attracting increasing research attention. Brevibacteria are the only bacteria of dairy significance that produce carotenoides, some of which can be aromatic (97). The metabolic capabilities of

B. linens, which include the production of sulphur compounds and pigments, have a major impact on cheese flavor and appearance (98,99). Plasmids are rare in brevibacteria but are sometimes found in B. levins (100,101) and also B. lactofermentum (102), which is used commercially for the production of amino acids and enzymes (103). However, to date no phenotypes have been attributed to native plasmids in brevibacteria.

Bifidobacteria are increasingly important cultures in the diary industry because of their prominent role in probiotics. It differs from other dairy cultures in that glucose is metabolized exclusively by the fructose-6-phosphate shunt, which utilizes the enzyme fructose-6-phosphoketolase (F6PPK). This is therefore a frequent diagnostic marker for this group of bacteria. Members of the genus Bifidobacterium are dominant flora in the large intestine of humans and also in the GI tracts of many farm animals. Studies throughout the twentieth century have consistently substantiated the association of good intestinal health with high bifidobacteria numbers (reviewed, 14). Because of this, bifidobacteria are important probiotic bacteria for humans (104) and farm animals (105), second only to lac-tobacilli in commercial dominance. The growing interest in bifidobacteria probiotics has greatly increased the research attention of these intriguing bacteria. The species of Bifidobacterium of relevance to the dairy industry include B. longum, B. infantis, B. bifidum, B. adolescentis, B. breve, and B. lactis. The latter species is a commonly used probiotic and it is intriguing that it is not a normal human inhabitant. It was first isolated in 1997 from fermented milk by Meile et al. (106) and was noted to have a higher tolerance to oxygen than other bifidobacteria. While it was genetically very close to B. animalis, it was deemed to have changed sufficiently during adaptation to fermentation environments to warrant a new species name. While the name B. lactis is a much more attractive name for a dairy culture, some studies question the justification for a new species name (107,108). The changes that occurred in B. lactis during its adaptation to fermentation conditions make it a very resilient strain that can remain viable during processing and storage, longer than other bifidobacteria. These practical reasons contribute to its popularity, but may limit its probiotic properties in the intestine. Plasmids are only found in some species of Bifidobacterium, being most common in B. longum followed by B. breve (109,110,111). It is interesting that most strains of B. longum will harbor plasmids, while B. infantis, which is very closely related to B. longum, does not harbor any plasmids. Plasmids are generally small (< 10 kb) and cryptic. A number have now being characterized at the sequence level (112,113,114). However, phenotypes outside of normal plasmid functions have not been found on bifidobacteria plasmids. One exception is a report of a plasmid linked to production of the bifidocin B bacteriocin in B. bifidum (115). It was interesting that the production of bifidocin B was linked to an ~8.0 kb plasmid, while the immunity did not appear to be correlated with it. As bacteriocin production and immunity genes are inherently linked together in bacteria, this would appear to be an unusual setup. However, sequence analysis of the plasmid will be needed to investigate this further.

10.3.3 Plasmid Replication in Dairy Cultures

The need for molecular understanding and genetic manipulation of starter culture pheno-types has resulted in extensive research into plasmid replication mechanisms in these bacteria. Understanding plasmid replication is essential for the development and application of molecular tools. The type of plasmid replication mechanism can influence many crucial aspects of plasmids, including structural and segregational stability (116). While bacterial plasmids are generally circular entities with the cell, some are present in both prokaryotes and eukaryotes that are linear (reviewed, 117). In the dairy cultures, the plasmids found have been invariably circular except for the report of a 48.5 kb linear plasmid in a Lactobacillus gasseri strain (118), which has not yet been characterized. Two modes of circular plasmid replication predominate in dairy cultures, rolling circle replication (RCR) and theta replication, with the former being much more common.

The primary distinguishing feature of RCR is the presence of single stranded DNA intermediates that accumulate during replication (116). RCR has been observed for all bacterial genera that are relevant to dairy cultures. The replication origins from RCR plasmids are conserved and depend on the presence of a replication initiation gene (rep). This gene encodes a Rep protein that initiates plasmid replication by nicking the DNA at the replication origin, which is a double strand origin termed ori, thus permitting replication of one plasmid DNA strand to begin, which is referred to as the leading strand. It also has a ligation role during replication and nicking roles during each subsequent round of replication. The single strand intermediates can be readily seen during replication and can be degraded by S1 nuclease, which is the procedure used to confirm RCR replication in these bacteria. The efficient conversion of single strand DNA into double stranded plasmid DNA is critical for the structural and segregational stability of the plasmid. This is initiated at a single strand origin (sso) that permits replication of this strand. Single strand origins tend to form extensive secondary structures, and this can influence plasmid stability. While plasmids utilizing RCR are very common among this group on organisms, and are used frequently as vectors for cloning and other molecular applications, they tend not to be very stable, both structurally and also segregationally. It should be noted that not all RCR plasmids suffer these defects to the same degree, as some are more efficient at converting the single strand intermediates into double strand plasmid DNA, via the sso, thus improving their stability. The sso therefore has a major role on plasmid stability during replication of RCR plasmids.

The other mode of plasmid replication that has been found among dairy cultures is theta replication, so named after the theta appearance of the plasmid during replication. The absence of single stranded intermediates during replication of these plasmids greatly increases their stability over RCR plasmids in general, and they are therefore more useful as cloning vectors. During theta plasmid replication, DNA nicking is not involved, but an RNA primer is synthesized that can initiate replication of both strands following denaturing the two strands around the origin. This opening of the two DNA strands around the origin resembles the theta symbol. While theta replication is very common for plasmids in gram negative bacteria it is much less frequently observed in gram positives. However, plasmids replicating by this mechanism have been observed, by functional analysis or sequence similarities, for many of the dairy related cultures, including Lactococcus (119,120), Lactobacillus (121), Enterococcus (122), Streptococcus thermophilus (123), Leuconostoc (124), Brevibacterium (101), and Propionibacterium (125).

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