Use of green fluorescent protein to monitor Lactobacillus sakei in fermented meat products

Gory, L

doi:10.1016/s0378-1097(00)00515-2

Cited by 24 publications

(40 citation statements)

References 22 publications

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“…The L. agilis La3 ldhL promoter produced the most efficient expression of the heterologous reporter gene gfp within both La3 and Lv5. Our results are consistent with other studies that have successfully utilized ldhL promoters to drive heterologous expression of the gfp gene within lactobacilli (20,26,56). There are exciting applications in using these strains to deliver proteins to the chicken GIT: either GFP, which could facilitate studies into the mechanism(s) of colonization (34,56), or therapeutic peptides, such as antigens to be used as mucosal vaccines (54), bacteriocins (38) as an alternative to antibiotics (22), or enzymes to increase production efficiency (25,42).…”

Section: Discussionsupporting

confidence: 92%

“…Native regulatory systems naturally inducible in vivo, or constitutive promoters, could ensure constant production of therapeutic peptides at the site of colonization. Several constitutive promoters have previously been used for heterologous expression in lactobacilli, including the P ldhL (20,26), P slpA (7,26,29,31), P 144 (15), and lactococcal P 23 (10) promoters. Recently, several groups have described the use of homologous promoters for highly efficient gene expression (11,16,30).…”

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confidence: 99%

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Transformation of, and Heterologous Protein Expression in, Lactobacillus agilis and Lactobacillus vaginalis Isolates from the Chicken Gastrointestinal Tract

Stephenson

Moore

Allison

2011

Appl Environ Microbiol

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Lactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous strains for the delivery of therapeutic proteins. Previously we identified three chicken-derived Lactobacillus strains, Lactobacillus agilis La3, Lactobacillus vaginalis Lv5, and Lactobacillus crispatus Lc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies of 10 8 and 10 6 transformants per microgram of plasmid DNA, respectively. The third strain tested, L. crispatus Lc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over 100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter gene gfp was analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3 ldhL promoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous protein expression in L. agilis and L. vaginalis. The ability of these strains to express heterologous proteins in vitro indicates their potential utility as in vivo delivery vectors for therapeutic peptides to the chicken gastrointestinal tract.Lactobacilli are autochthonous inhabitants of the chicken gastrointestinal tract (GIT), where they predominate the upper GIT microbiota (53). Their dominance of the upper GIT makes lactobacilli excellent candidates for development as live vectors for the delivery of therapeutic proteins targeting bacterial pathogens such as Clostridium perfringens, Campylobacter jejuni, Listeria monocytogenes, or Salmonella enterica. Additionally, lactobacillus delivery vectors could be used to immunize against avian viruses, such as infectious bursal disease, Marek's disease, Newcastle disease, or avian influenza virus. We have recently identified several Lactobacillus strains, Lactobacillus agilis La3, Lactobacillus vaginalis Lv5, and Lactobacillus crispatus Lc9, which are able to colonize and persist within the GIT of chickens fed either a commercial or a highprotein diet (45). These strains show great potential as vectors for the in situ delivery of therapeutic proteins targeting GIT pathogens.Lactobacilli have several advantages as mucosal delivery vectors (54), including "generally regarded as safe" status, survival within the GIT, stimulation of immune responses in the mucosa, and the potential to be engineered to express therapeutic peptides. In regard to the latter, lactobacilli are notoriously recalcitrant to transformation, representing an important limitation in realizing the vectoring potential of persistent strains.Several studies have investigated the genetic transformation of chicken Lactobac...

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Section: Discussionsupporting

confidence: 92%

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confidence: 99%

Transformation of, and Heterologous Protein Expression in, Lactobacillus agilis and Lactobacillus vaginalis Isolates from the Chicken Gastrointestinal Tract

Stephenson

Moore

Allison

2011

Appl Environ Microbiol

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“…cremoris MG1363 (38) carrying plasmid pJIM2246:gfp was provided by Marie-Pierre Chapot-Chartier (Micalis, INRA, Jouy-en-Josas, France). Briefly, it was obtained by transferring the transcriptional fusion of the ldhL promoter with gfp (described by Gory et al [13]) in the pJIM2246 vector (28). This allows the constitutive expression of GFP under the transcriptional control of the Lactobacillus sakei promoter of the lactate dehydrogenase gene (ldh).…”

Section: Methodsmentioning

confidence: 99%

Spatial Distribution of Bacterial Colonies in a Model Cheese

Jeanson

Chadœuf

Madec

et al. 2011

Appl Environ Microbiol

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In most ripened cheeses, bacteria are responsible for the ripening process. Immobilized in the cheese matrix, they grow as colonies. Therefore, their distribution as well as the distance between them are of major importance for ripening steps since metabolites diffuse within the cheese matrix. No data are available to date about the spatial distribution of bacterial colonies in cheese. This is the first study to model the distribution of bacterial colonies in a food-type matrix using nondestructive techniques. We compared (i) the mean theoretical three-dimensional (3D) distances between colonies calculated on the basis of inoculation levels and considering colony distribution to be random and (ii) experimental measurements using confocal microscopy photographs of fluorescent colonies of a Lactococcus lactis strain producing green fluorescent protein (GFP) inoculated, at different levels, into a model cheese made by ultrafiltration (UF). Enumerations showed that the final numbers of cells were identical whatever the inoculation level (10 4 to 10 7 CFU/g). Bacterial colonies were shown to be randomly distributed, fitting Poisson's model. The initial inoculation level strongly influenced the mean distances between colonies (from 25 m to 250 m) and also their mean diameters. The lower the inoculation level, the larger the colonies were and the further away from each other. Multiplying the inoculation level by 50 multiplied the interfacial area of exchange with the cheese matrix by 7 for the same cell biomass. We finally suggested that final cell numbers should be discussed together with inoculation levels to take into account the distribution and, consequently, the interfacial area of colonies, which can have a significant influence on the cheese-ripening process on a microscopic scale.During cheese making, regardless of the cheese type, bacteria are immobilized in the curd during the coagulation step. It is generally accepted that 90% of the bacteria present in the milk are retained, trapped in the curd, while only 10% are lost in the whey during draining (16). In cheeses made by ultrafiltration (UF), the draining step is absent, and 100% of the cells are then retained in the curd. In any case, after immobilization by coagulation, each inoculated bacterial cell is assumed to grow, generating a colony inside the curd. Colonies are then spread within the cheese curd, and they interact with the cheese matrix during ripening. Consequently, the ripening process must take place on a microscopic scale around colonies. Only studies showing microscopic examinations of bacterial colonies in cheese either by electronic microscopy (24) or, more recently, by confocal laser scanning microscopy (7, 19) have been reported.The ripening process (proteolysis, lipolysis, amino acid catabolism, and the production of organic acids, etc.) relies on the metabolic activities of bacterial colonies, leading to the formation of flavors and textures of cheese (11,25). So far, ripening has always been described with average processes on the che...

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“…For example, pG + host derivatives are not thermosensitive in Lactobacillus sakei (Gory et al, 2001) and Lactobacillus plantarum (strain CCM 1904;P. Horvath & B. Kammerer, personal communication).…”

Section: Discussionmentioning

confidence: 99%

Transposition in Lactobacillus delbrueckii subsp. bulgaricus: identification of two thermosensitive replicons and two functional insertion sequences

et al. 2003

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Use of green fluorescent protein to monitor Lactobacillus sakei in fermented meat products

Cited by 24 publications

References 22 publications

Transformation of, and Heterologous Protein Expression in, Lactobacillus agilis and Lactobacillus vaginalis Isolates from the Chicken Gastrointestinal Tract

Transformation of, and Heterologous Protein Expression in, Lactobacillus agilis and Lactobacillus vaginalis Isolates from the Chicken Gastrointestinal Tract

Spatial Distribution of Bacterial Colonies in a Model Cheese

Transposition in Lactobacillus delbrueckii subsp. bulgaricus: identification of two thermosensitive replicons and two functional insertion sequences

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