Lactococcus lactis
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| Lactococcus lactis |
Lactococcus lactis is a non pathogenic, gram-positive bacteria known as one of the most important microbes in dairy food production. It is closely related to the streptococcus genus and has two subspecies, L. lactis subsp. and L. cremoris subsp. They are used to make soft and hard cheese respectively. L. Lactis group in pairs or short chains with a length of about 1.5µm. Lactococcus are immotile, nonsporelating, and are usually spherical or ovoid cells.
L. Lactis is the starter culture for the production of fermented dairy products such as milk, cheese and yogurt. Lactococcus is considered a opportunistic pathogen, because of its ability to convert almost all of its sugar into lactic acid. Lactic acid produced by the the microbe curdles the milk which separates into curds and in turn is used to produce cheese and whey.
In nature, L. lactis is found to be inactive on plant surfaces and on to multiply in the gastrointestinal tract after being swallowed by an animal.
Genome structure
L. lactis' genome has been sequenced to investigate, what specificly, is its essential role in dairy fermentation. Unexpected features of the analyzed genome have been disclosed, genes are seen to enable the bacterium to perform aerobic respiration along with horizontal gene transfer by transformation. Decades of research has been attributed to the genome sequencing and comparative genomics of L. lactis to unveil the characteristics of L.lactis that can specifically develop flavor, and improve quality and preservation of dairy products such as cheese.
L. lactis is a circular chromosome, its genome contains 2,365,589 base pairs. Protein-coding genes represent 86% of the genome, stable RNA 1.4%, and noncoding regions 12.6%. These percentages are very similar to other bacteria genome. Biological functions have been assigned to 64% of the genome, 20.1% have similar genome to other unknown proteins, however, 15% of the genome is unidentifiable. This 15% is hypothesized to contain the traits specific to L.lactis.
Cell structure and metabolism
"Lactococcus lactis" can function in both aerobic and anaerobic environments. The bacterium's primitive source of energy is produced anaerobically, which results in the accumulation of lactic acid. The deprivation of oxygen leads the glycolysis process to breakdown carbohydrates into pyruvate which then converts into lactic acid. This process is only possible through the production of the lactate dehydrogenase enzyme and NAD. Lactate is transported to the median which causes the efflux of protons, resulting in the appropriate membrane potential for energy production. Some strains of L. lactis are capable of growing under aerobic conditions, when oxygen and a heme source is present new traits are observed, such as increased growth index, resistance to oxidative and acid stress, and long-termed endurance at low temperatures. Along with the heme source, the presence of NADH-oxidases has been liked to aerobic respiration, which has shown increased cell growth and production of proteins and vitamins.
Application to Biotechnology
Extensive research has been attributed to the sequencing of Lactococcus lactis' genome due to its major role in the production of dairy products and preservation. Manufactures use the discovered properties of L.lactis to increase food preservation, distinguish flavor and aroma. L.lactis contains a bacteriocin, a natural antimocrobial agent that fights against a wide range of Gram-positive bacteria, such as food-bourne pathogens. Uses of nisin to control spoilage lactic acid bacteria have been identified in beer, wine, alcohol production, and high acid foods such as salad dressings.
A recently discovered application of Lactococcus lactis is in the development of vaccine delivery systems. L.lactis can be genetically engineered to generate proteins from pathogenic species on their cell surfaces. Mucosal administration of the modified strain will induce an immune response to the cloned protein and provide immunity to the pathogen. Mucosal immunity is a main concern of the public health since it is the primary way of pathogenic entry. In underdeveloped countries, where diseases spread rapidly, mucosal immunity can facilitate the distribution of vaccines since it less cost effective and easily administered. This approach theoretically can be applied to any pathogen that enters a human or mammal through a mucoasal surface, however, it is most commonly used to provide immunity to Streptococcus pyogenes, the pathogenic agent of strep throat.
Economical Importance
L.Lactis holds great value to the dairy product industry. The bacteria is found in milk and its main function is to produce lactic acid, which improves preservation of the 10 million pounds of cheese produced annually. The bacterium can be a single strain starter culture or a mixed strain culture with other lactic acid bacteria. It is a key starter culture in the production of cheese's such as cheddar, colby, cottage cheese, cream cheese, etc. as well as other dairy products like cultured butter, buttermilk, and sour cream. The cheese producing industry has contributed a great deal to the economy. In Wisconsin alone 2.5 billion lbs of cheese are produced and 90% of their milk is used to producing cheese. Wisconsin's cheese producing revenue totals up to $18 billion. The cheese industry is of such great importance in Wisconsin that it has nominated Lactococcus lactis as it's state microbe.
Current Research
Immunogenicity and protective efficacy of orally administered recombinant Lactococcus lactis expressing surface-bound HIV Env
The use of L.lactis in successful vaccine delivery has encouraged many researchers to further investigate the various pathogens this approach can be appllied to. This study applies this theory to the human immunodeficiency virus (HIV). Researchers created a recombinant vector using L.lactis, the newly engineered L.lactis expressed the Env gene of HIV, specifically, the V2-V4 loop of HIV Env. Mice were then fed the vector containing HIV, along with a control group who were fed a vector with a HIV Env–expressing vaccinia virus. Results showed that the HIV vaccinated mice obtained a viral load 350 times less than the control group. This outcome has encouraged the further development of a L. lactis based HIV vaccine.
Innate inflammatory responses to the Gram-positive bacterium Lactococcus lactis
The impressive use of L.lactis as a useful immunity tool has promoted further research to support the potential applications of the bacteria. In this study L.lactis' adjuvant characteristics are comparatively investigated with other bacteria. The study contained comparative data on the proinflammatory effects of L. lactis strain NZ9000, a non-pathogenic bacterium, with E. coli strain DH5α and Salmonella typhi strain Ty21a, both non-pathogenic strains of pathogenic bacteria. Results showed that when L.lactis, E.coli, and S.typhi were co-incubated with B10R murine macrophages, in vitro, they all expressed pro-inflammatory properties. However, L.lactis expressed lower levels of chemokine mRNA expression. Leukocyte recruitment was also compared, in vivo, between all three bacteriums. L. lactis, E. coli and S. typhi showed similar levels of leukocyte recruitment into murine air-pouches, these recruited cells displayed a specific activation status according to the bacterial stimuli. The results demonstate L. lactis' ability to induce chemokine expression both in vitro and in vivo, and displays similar potency as S. typhi, an established live vaccine.
A new plasmid vector for DNA delivery using lactococci
Continuous studies are done trying to improve the use of L.lactis as a DNA carrier for such uses as mucosal vaccines. In this study researchers strive to develop a new type of plasmid that can be used with L.lactis in antigen transfer. The new plasmid,pValac, was created by fusing a prokaryotic and eukaryotic region together. The gfp ORF was cloned into the pValac and was analysed by transfection in PK15 cells. Pvalac:gfp's potential was tested by combining it with Lactococcus lactis inlA+ strains and attempting to insert it into Caco-2 cells. Results showed that after transfection with pValac:gfp, GFP expression was observed in PK15 cells. L. lactis inlA+ were able to invade Caco-2 cells and delivered a functional expression cassette (pCMV:gfp) into epithelial cells.