| Lactococcus lactis|
Lactococcus lactis is a non-pathogenic, Gram-positive bacteria, known for its essential role 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 is a well-studied bacterium due to its contribution to the dairy food and pharmaceutical industries.
L. lactis is the starter culture for the production of fermented dairy products such as milk, cheese and yogurt. Lactococcus attains the ability to convert almost all of its sugar into lactic acid. Lactic acid produced by the microbe curdles the milk, which separates into curds and in turn is used to produce cheese. The most recent application of L. lactis is in the delivery of vaccines.
L. lactis' genome has been sequenced to investigate, what specifically, is its essential role in dairy fermentation. Unexpected features of the analyzed genome have been disclosed; including genes that 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. Eighty-six percent of the genome can be attributed to protein-coding genes, 1.4% is stable RNA, and non-coding regions 12.6%. These percentages are very similar to other bacteria genome. Biological functions have been assigned to 64% of the genome, 20.1% encode similar proteins found in other genomes, and 15% of the genome is unidentifiable. This 15% is hypothesized to contain the traits specific to L. lactis.
Cell structure and metabolism
L. lactis is a Gram-negative, spherical shaped bacterium. The microbe may group in pairs or in short chains, with a length of about 1.5 µm. Lactococcus are immotile, nonsporelating, and are usually spherical or ovoid cells.
Lactococcus lactis can function in both aerobic and anaerobic environments. The bacterium's primary 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, and ultimately 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. The oxygen and a heme source present new traits, 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 linked to aerobic respiration, which has shown increased cell growth and production of proteins and vitamins. Due to L. lactis inadequate biosynthetic ability, it requires an external source of fermentable sugar, vitamins, potassium, magnesium, and amino acids to grow favorably.
Lactococcus lactis is an opportunistic bacteria, in nature, it can be found on wild plants and consequently in the gastrointestinal tract of animals, such as cows, and are ultimately inoculated in milk. A study has been done showing the survival rate of ingested L. lactis. Data has shown that only 2% of all the ingested bacteria is found in the remaining feces if consumed within three days.
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, called nisin. It is a natural antimicrobial agent that fights against a wide range of Gram-positive bacteria, such as food-borne pathogens. Uses of nisin, such as controlling spoilage in lactic acid bacteria, have been present in alcohol production, wine, beer, and high acidic foods such as salad dressings.
The most recent 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 replicated 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 is less cost effective and easily administered. This approach theoretically can be applied to any pathogen that enters a human or mammal through a mucosal surface; however, it is most commonly used to provide immunity to Streptococcus pyogenes, the pathogenic agent of strep throat.
L. lactis holds great value to the dairy product industry. The bacterium is found in milk and its main function is to produce lactic acid, which improves preservation needed for over nine million pounds of cheese produced each year. 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 varies types of cheese such as cheddar, colby, cottage cheese, cream cheese, as well as other dairy products like cultured butter, buttermilk, and sour cream. The cheese producing industry has contributed a great deal to the U.S. economy. In Wisconsin alone 2.5 billion lbs of cheese are produced annually and 90% of their milk is used to produce cheese. Wisconsin's cheese producing revenue has accumulated up to $18 billion a year. The cheese industry is of such great importance in Wisconsin, that it has nominated Lactococcus lactis as it's state microbe.
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 applied 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 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. typhus 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 bacterium. 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 demonstrate L. lactis' ability to induce chemokine expression both in vitro and in vivo, and displays similar potency as S. typhi, an already established live vaccine.
A new plasmid vector for DNA delivery using lactococci
Continuous studies are constantly emerging 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. Fusing a prokaryotic and eukaryotic region together created the new plasmid, pValac. The gfp ORF was cloned into the pValac and was analyzed by transfection in PK15 cells. Pvalac:gfp's potential was tested by combining it with Lactococcus lactis in lA+ 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 in lA+ was successful in entering Caco-2 cells and upon entering, conveyed functional expression cassette (pCMV:gfp) into epithelial cells.
- Alexander Bolotin, Patrick Wincker, Stéphane Mauger, Olivier Jaillon, Karine Malarme, Jean Weissenbach, S. Dusko Ehrlich, and Alexei Sorokin. “The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403”. Cold Spring Harbor Laboratory Press. 2001. Volume 11. Issue 5. p. 731-753.
- Hols, P., Kleerebezem, M., Schanck, A., Ferain, T., Hugenholtz, J., Delcour, J., and de Vos, W.M. “Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering”. Nature Biotechnology. 1999. Volume 17. p. 588-592.
- Brooijmans, R.J.W., Poolman, B., Schuurman-Wolters, G.K., de Vos, W.M., and Hugenholtz, J. “Generation of membrane potential by Lactococcus lactis through aerobic electron transport”. Journal of Bacteriology. 2007.
- Xin KQ., Hoshino, Y., Toda, Y., Igimi, S., Kojima, Y., Jounai, N., Ohba, K., Kushiro, A., Kiwaki, M., Hamajima, K., Klinman, D., Okuda, K. "Immunogenicity and protective efficacy of orally administered recombinant Lactococcus lactis expressing surface-bound HIV Env". Blood. 2003 Jul 1;102(1):223-8. Epub 2003 Mar 20.
- Yama, K., Pouliota, P., N’diayea, M., Fourniera, S., Oliviera, M., and Cousineaua, B. "Innate inflammatory responses to the Gram-positive bacterium Lactococcus lactis" Vaccine. Volume 26, Issue 22, 23 May 2008, Pages 2689-2699
- Guimarães, V., Innocenti, S., Chatel, J., Lefèvre, J., Langella, P., Azevedo, V., and Miyoshi, A. "A new plasmid vector for DNA delivery using lactococci" Genetic Vaccines and Therapy. February, 2009. doi:10.1186/1479-0556-7-4