scholarly journals Introduction of Modified BglBrick System in Lactococcus lactis for Straightforward Assembly of Multiple Gene Cassettes

2021 ◽  
Vol 9 ◽  
Author(s):  
Tina Vida Plavec ◽  
Tim Ključevšek ◽  
Aleš Berlec
Keyword(s):  
Lactic Acid ◽  
Lactococcus Lactis ◽  
Fluorescent Proteins ◽  
Modular Construction ◽  
Transcription Terminator ◽  
Bacterial Surface ◽  
Protein Encoding ◽  
Intracellular Expression ◽  
Relevant Field ◽  
Protein Binders

Genetic modification of lactic acid bacteria is an evolving and highly relevant field of research that allows the engineered bacteria to be equipped with the desired functions through the controlled expression of the recombinant protein. Novel genetic engineering techniques offer the advantage of being faster, easier and more efficient in incorporating modifications to the original bacterial strain. Here, we have developed a modified BglBrick system, originally introduced in Escherichia coli and optimized it for the lactic acid bacterium Lactococcus lactis. Six different expression cassettes, encoding model proteins, were assembled in different order as parts of a modified BglBrick system in a novel plasmid pNBBX. All cassettes included nisin promoter, protein encoding gene and transcription terminator. We demonstrated successful intracellular expression of the two fluorescent proteins and display of the four protein binders on the bacterial surface. These were expressed either alone or concomitantly, in combinations of three model proteins. Thus, a modified BglBrick system developed herein enables simple and modular construction of multigene plasmids and controlled simultaneous expression of three proteins in L. lactis.

scholarly journals Riboflavin Production in Lactococcus lactis: Potential for In Situ Production of Vitamin-Enriched Foods

2004 ◽  
Vol 70 (10) ◽  
pp. 5769-5777 ◽  
Author(s):  
Catherine Burgess ◽  
Mary O'Connell-Motherway ◽  
Wilbert Sybesma ◽  
Jeroen Hugenholtz ◽  
Douwe van Sinderen
Keyword(s):  
Functional Analysis ◽  
Lactic Acid ◽  
Lactococcus Lactis ◽  
Regulatory Region ◽  
Northern Hybridization ◽  
Vitamin B ◽  
Fermented Foods ◽  
Riboflavin Production ◽  
In Situ Production

ABSTRACT This study describes the genetic analysis of the riboflavin (vitamin B2) biosynthetic (rib) operon in the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NZ9000. Functional analysis of the genes of the L. lactis rib operon was performed by using complementation studies, as well as by deletion analysis. In addition, gene-specific genetic engineering was used to examine which genes of the rib operon need to be overexpressed in order to effect riboflavin overproduction. Transcriptional regulation of the L. lactis riboflavin biosynthetic process was investigated by using Northern hybridization and primer extension, as well as the analysis of roseoflavin-induced riboflavin-overproducing L. lactis isolates. The latter analysis revealed the presence of both nucleotide replacements and deletions in the regulatory region of the rib operon. The results presented here are an important step toward the development of fermented foods containing increased levels of riboflavin, produced in situ, thus negating the need for vitamin fortification.

On the Microbiological Profile of Traditional Portuguese Sourdough

1999 ◽  
Vol 62 (12) ◽  
pp. 1416-1429 ◽  
Author(s):  
J. MIGUEL ROCHA ◽  
F. XAVIER MALCATA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactococcus Lactis ◽  
Dominant Species ◽  
Geographic Locations ◽  
Microbiological Profile ◽  
Enterococcus Casseliflavus ◽  
Lactobacillus Spp

Traditional manufacture of bread from maize has been noted to play important roles from both economic and social standpoints; however, enforcement of increasingly strict hygiene standards requires thorough knowledge of the adventitious microbiota of the departing dough. To this goal, sourdough as well as maize and rye flours from several geographic locations and in two different periods within the agricultural year were assayed for their microbiota in sequential steps of quantification and identification. More than 400 strains were isolated and taxonomic differentiation between them was via Biomerieux API galleries (375 of which were successfully identified) following preliminary biochemical and morphological screening. The dominant groups were yeasts and lactic acid bacteria (LAB). The most frequently isolated yeasts were Saccharomyces cerevisiae and Candida pelliculosa. The most frequently isolated LAB were (heterofermentative) Leuconostoc spp. and (homo-fermentative) Lactobacillus spp.; L. brevis, L. curvatus, and L. lactis ssp. lactis were the dominant species for the Lactobacillus genera; Lactococcus lactis ssp. lactis for lactococci; Enterococcus casseliflavus, E. durans, and E. faecium for enterococci; and Streptococcus constellantus and S. equinus for streptococci.

scholarly journals EFFECT OF EXOPOLISACCHARIDES OF LACTIC ACID BACTERIA ON THE SYNTHESIS OF PROINFLAMMATORY CYTOKINES BY MACROPHAGIC MICE IN PHAGOCYTOSIS OF STAPHYLOCOCCUS AUREUS

2018 ◽  
pp. 67-71
Author(s):  
G. T. Uryadova ◽  
E. A. Gorelnikova ◽  
N. A. Fokina ◽  
A. S. Dolmashkina ◽  
L. V. Karpunina
Keyword(s):  
Staphylococcus Aureus ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactococcus Lactis ◽  
Inflammatory Cytokines ◽  
Proinflammatory Cytokines ◽  
Streptococcus Thermophilus ◽  
Ambiguous Effect ◽  
Pro Inflammatory Cytokines

Aim. Study of the effect of exopolysaccharides (EPS) of lactic acid cocci on cytokine activity of macrophages of mice with phagocytosis in vitro Staphylococcus aureus 209-P. Materials and methods. The EPS of Streptococcus thermophilus and Lactococcus lactis B-1662 was used in the work. At 13, 5 and 7, AMP and PMP were isolated and the phagocytosis process was modeled in vitro. After 30 minutes, 1, 6 and 24 hours, the content of pro-inflammatory cytokines IL-1a and TNF-a was determined. Results. EPSs had an ambiguous effect on the production of cytokines. The greatest effect on the synthesis was provided by EPS of S. thermophilus. Conclusion. The results of the study allow us to talk about the possibility of using EPS of S. thermophilus as a preventive immunomodulator for correction of the cytokine status of animals.

scholarly journals A SH3_5 Cell Anchoring Domain for Non-recombinant Surface Display on Lactic Acid Bacteria

2021 ◽  
Vol 8 ◽  
Author(s):  
Pei Kun Richie Tay ◽  
Pei Yu Lim ◽  
Dave Siak-Wei Ow
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Surface Display ◽  
Bioactive Molecules ◽  
Gastric Digestion ◽  
Functional Protein ◽  
Fluorescent Reporter ◽  
Surface Conditions ◽  
Bacterial Surface

Lactic acid bacteria (LAB) are a group of gut commensals increasingly recognized for their potential to deliver bioactive molecules in vivo. The delivery of therapeutic proteins, in particular, can be achieved by anchoring them to the bacterial surface, and various anchoring domains have been described for this application. Here, we investigated a new cell anchoring domain (CAD4a) isolated from a Lactobacillus protein, containing repeats of a SH3_5 motif that binds non-covalently to peptidoglycan in the LAB cell wall. Using a fluorescent reporter, we showed that C-terminal CAD4a bound Lactobacillus fermentum selectively out of a panel of LAB strains, and cell anchoring was uniform across the cell surface. Conditions affecting CAD4a anchoring were studied, including temperature, pH, salt concentration, and bacterial growth phase. Quantitative analysis showed that CAD4a allowed display of 105 molecules of monomeric protein per cell. We demonstrated the surface display of a functional protein with superoxide dismutase (SOD), an antioxidant enzyme potentially useful for treating gut inflammation. SOD displayed on cells could be protected from gastric digestion using a polymer matrix. Taken together, our results show the feasibility of using CAD4a as a novel cell anchor for protein surface display on LAB.

scholarly journals Genome Sequences of Lactococcus lactis MG1363 (Revised) and NZ9000 and Comparative Physiological Studies

2010 ◽  
Vol 192 (21) ◽  
pp. 5806-5812 ◽  
Author(s):  
Daniel M. Linares ◽  
Jan Kok ◽  
Bert Poolman
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Glucose Uptake ◽  
Lactococcus Lactis ◽  
Point Mutations ◽  
Genome Sequences ◽  
Sugar Fermentation ◽  
Sequencing Strategy ◽  
Physiological Studies ◽  
Transcriptomic Studies

ABSTRACT Lactococcus lactis NZ9000 and its parent MG1363 are the most commonly used lactic acid bacteria for expression and physiological studies. We noted unexpected but significant differences in the growth behaviors of both strains. We sequenced the entire genomes of the original NZ9000 and MG1363 strains using an ultradeep sequencing strategy. The analysis of the L. lactis NZ9000 genome yielded 79 differences, mostly point mutations, with the annotated genome sequence of L. lactis MG1363. Resequencing of the MG1363 strain revealed that 73 out of the 79 differences were due to errors in the published sequence. Comparative transcriptomic studies revealed several differences in the regulation of genes involved in sugar fermentation, which can be explained by two specific mutations in a region of the ptcC promoter with a key role in the regulation of cellobiose and glucose uptake.

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