Chromosomal integration of the green fluorescent protein gene in lactic acid bacteria and the survival of marked strains in human gut simulations

Scott, Karen P.; Mercer, Derry K.; Richardson, Anthony J.; Melville, Claire M.; Glover, Lesley Anne; Flint, Harry J.

doi:10.1111/j.1574-6968.2000.tb08867.x

Cited by 42 publications

(7 citation statements)

References 21 publications

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“…Several other studies have reported on the successful use of homologous recombination for the stable integration of genes in bacterial genomes (38,49,50). Our results indicated that disruption or inactivation of the chromosomally located L. plantarum 423 dam gene was not lethal and had no impact on the strain's growth capacity.…”

Section: Discussionsupporting

confidence: 57%

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

Zyl

Deane

Dicks

2015

Appl Environ Microbiol

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Lactic acid bacteria (LAB) are natural inhabitants of the gastrointestinal tract (GIT) of humans and animals, and some LAB species receive considerable attention due to their health benefits. Although many papers have been published on probiotic LAB, only a few reports have been published on the migration and colonization of the cells in the GIT. This is due mostly to the lack of efficient reporter systems. In this study, we report on the application of the fluorescent mCherry protein in the in vivo tagging of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423. The mCherry gene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome of L. plantarum 423 by homologous recombination. In the case of E. mundtii ST4SA, the mCherry gene was cloned into the pGKV223D LAB/Escherichia coli expression vector. Expression of the mCherry gene did not alter the growth rate of the two strains and had no effect on bacteriocin production. Both strains colonized the cecum and colon of mice. Lactic acid bacteria (LAB) are common inhabitants of a healthy human gastrointestinal tract (GIT). Lactobacillus spp. are used as starter cultures in many fermented foods and are well known for their probiotic properties and the exclusion of pathogens from the GIT (1-6). Some strains have been closely associated with the treatment of gastrointestinal disorders, lactose intolerance, and stimulation of the immune system (7,8).Despite the increasing consumer interest in probiotic LAB, the mechanisms whereby these bacteria exert their beneficial effects in the GIT are not well understood (9). Although simulated gastrointestinal models (10-14) produced valuable data on the colonization of probiotic LAB and the exclusion of pathogens, the method remains an in vitro approach and is not a true reflection of in situ conditions. Labeling of probiotic bacteria with genes expressing fluorophores allows studying colonization and competition between gut microorganisms in vivo (15)(16)(17)(18)(19).Since the discovery of the green fluorescent protein (GFP) by Shimomura et al. (20), a number of genetic variants that emit light at longer wavelengths, and that are more suited for in vivo animal studies, have been described (21-23). The mCherry red fluorescence protein (RFP), a variant of the Discosoma red (DsRed) protein (24,25), is excited at wavelengths longer than 600 nm and is photostable (26-28). Studies conducted with Bacillus subtilis (29) and Escherichia coli (30) showed that expression of the mCherry protein, even at high levels, had no effect on the cell's physiology. Tauer and coworkers (31) used the mCherry gene to study recombinant protein expression in Lactobacillus plantarum. Mohedano and coworkers (32) adapted the broad-host-range vector pNZ8048 to study gene expression in Lactobacillus and showed that the mCherry construct could be used to study complex promoter induction mechanisms, such as bacteriocin production by Lactobacillus acidophilus. The role that lactococcin 972 (...

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Section: Discussionsupporting

confidence: 57%

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

Zyl

Deane

Dicks

2015

Appl Environ Microbiol

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“…However, imprecise repair of DSBs has the potential to be highly deleterious, as it can lead to genome instability, including the formation of chromosomal rearrangements. In particular, chromosomal translocations can arise when DNA ends from DSBs on two heterologous chromosomes are improperly joined (Scott et al, 2000). Given this, researchers have been taking advantage of various nucleases, especially the recently developed programmable nucleases, to deliberately induce DSBs at loci of interest to generate translocations.…”

Section: Introductionmentioning

confidence: 99%

Induction of Chromosomal Translocations with CRISPR-Cas9 and Other Nucleases: Understanding the Repair Mechanisms That Give Rise to Translocations

Brunet

Jasin

2018

Advances in Experimental Medicine and Biology

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Chromosomal translocations are associated with several tumor types, including hematopoietic malignancies, sarcomas, and solid tumors of epithelial origin, due to their activation of a proto-oncogene or generation of a novel fusion protein with oncogenic potential. In many cases, the availability of suitable human models has been lacking because of the difficulty in recapitulating precise expression of the fusion protein or other reasons. Further, understanding how translocations form mechanistically has been a goal, as it may suggest ways to prevent their occurrence. Chromosomal translocations arise when DNA ends from double-strand breaks (DSBs) on two heterologous chromosomes are improperly joined. This review provides a summary of DSB repair mechanisms and their contribution to translocation formation, the various programmable nuclease platforms that have been used to generate translocations, and the successes that have been achieved in this area.

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“…Successful expression of GFP in different lactic acid bacteria has been reported from several laboratories [15] [16] [13]. The ability of streptococcal strains expressing GFP to survive in vivo and to be suitable for pathogenesis studies has been demonstrated for Streptococcus suis [15].…”

Section: Introductionmentioning

confidence: 99%

High-Level Fluorescence Labeling of Gram-Positive Pathogens

et al. 2011

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Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10–50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.

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Chromosomal integration of the green fluorescent protein gene in lactic acid bacteria and the survival of marked strains in human gut simulations

Cited by 42 publications

References 21 publications

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

Induction of Chromosomal Translocations with CRISPR-Cas9 and Other Nucleases: Understanding the Repair Mechanisms That Give Rise to Translocations

High-Level Fluorescence Labeling of Gram-Positive Pathogens

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