scholarly journals Heterologous Expression of the Lactococcus lactis Bacteriocin, Nisin, in a Dairy Enterococcus Strain

2002 ◽  
Vol 68 (7) ◽  
pp. 3392-3400 ◽  
Author(s):  
Haiping Li ◽  
Daniel J. O'Sullivan
Keyword(s):  
Signal Transduction ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Heterologous Expression ◽  
Lactococcus Lactis ◽  
Pcr Amplification ◽  
Northern Hybridization ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Enterococcus Strain

ABSTRACT The bacteriocin nisin is produced only by some strains of Lactococcus lactis, and to date production in other lactic acid bacteria has not been achieved. Enterococcus sp. strain N12β is a nisin-immune transconjugant obtained from a nisin-producing donor (L. lactis ATCC 11454) and a dairy recipient (Enterococcus sp. strain S12β), but it does not produce nisin. In this study, using PCR amplification, we confirmed that the whole nisin operon is likely present in Enterococcus sp. strain N12β. Northern hybridization of total RNA from strain N12β with a nisA probe and the results of reverse transcriptase PCR showed the lack of nisA transcription in this strain. However, nisA transcription was partially restored in strain N12β upon growth in the presence of exogenous nisin, and the nisA transcription signal was intensified after an increase in the external nisin level. Furthermore, bioassays showed that active nisin was produced in a dose-dependent fashion by strain N12β following induction by exogenous nisin. These results indicated that expression of the nisin genes in Enterococcus sp. strain N12β depended on autoinduction via signal transduction. However, the amount of external inducing signal required was significantly greater than the amount needed for autoinduction in L. lactis.

scholarly journals Isolation, identification and antibiotic susceptibility of nis+ Lactococcus lactis from dairy and non-dairy sources

2013 ◽  
Vol 31 (No. 4) ◽  
pp. 323-331 ◽  
Author(s):  
P. Khemariya ◽  
S. Singh ◽  
G. Nath ◽  
A.K. Gulati
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Antimicrobial Agent ◽  
Lactococcus Lactis ◽  
Antibiotic Susceptibility ◽  
Pcr Amplification ◽  
Food Borne ◽  
Antibiotic Susceptibility Test ◽  
Different Types ◽  
Zones Of Inhibition

Eight isolates of Lactococcus lactis subsp. lactis were isolated and identified by phenotypic and molecular characterisation out of 23 isolates of lactic acid bacteria (LAB) from different dairy and non-dairy sources. Out of eight strains, four were obtained from dairy and four from non-dairy sources. All eight strains of L. lactis subsp. lactis were able to produce zones of inhibition against the Lactobacillus acidophilus NCDC 015. The antimicrobial agent produced by the isolates inhibited the growth of a range of related lactic acid bacteria and certain Gram positive food-borne microorganisms. The antimicrobial agent, i.e. nisin, produced by the strains was confirmed by PCR amplification of nisin gene sequences of 174 bp size. Antibiotic susceptibility test to 21 different types of antibiotics was evaluated. All the isolates were resistant to fosfomycin, cefepime, amikacin, kanamycin, neomycin, nalidixic acid, pipemidic acid, norfloxacin, sulphadiazine, colistin, polymixin, teicoplanin, nystatin, and amphotericin B but susceptible to ampicillin, erythromycin, spiramycin, spectinomycin, ciprofloxacin, rifampicin, and trimethoprim.  

scholarly journals D-Alanyl-D-Alanine Ligase as a Broad-Host-Range Counterselection Marker in Vancomycin-Resistant Lactic Acid Bacteria

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Shenwei Zhang ◽  
Jee-Hwan Oh ◽  
Laura M. Alexander ◽  
Mustafa Özçam ◽  
Jan-Peter van Pijkeren
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Heterologous Expression ◽  
Genome Editing ◽  
Vancomycin Resistance ◽  
Dependent Manner ◽  
Content Type ◽  
Suicide Vector ◽  
Vancomycin Resistant ◽  
Dose Dependent

ABSTRACTThe peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending ind-alanyl-d-lactate and binds with high affinity to peptidoglycan ending ind-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a natived-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genusLactobacillus. Finally, our system represents the first “plug and play” counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCEThe genusLactobacilluscontains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selectedLactobacillusspecies. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)—an essential enzyme involved in peptidoglycan synthesis—increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance inLactobacillus; 81% ofLactobacillusspecies are intrinsically resistant to vancomycin, which makes our tool broadly applicable.

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 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.

scholarly journals Evaluation of the Efficiency of Ethanol Precipitation and Ultrafiltration on the Purification and Characteristics of Exopolysaccharides Produced by Three Lactic Acid Bacteria

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Manel Ziadi ◽  
Taroub Bouzaiene ◽  
Sana M’Hir ◽  
Kaouther Zaafouri ◽  
Ferid Mokhtar ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactobacillus Plantarum ◽  
Lactococcus Lactis ◽  
Leuconostoc Mesenteroides ◽  
Ftir Analysis ◽  
Molecular Weights ◽  
Molar Ratios ◽  
Monomer Composition ◽  
Ethanol Precipitation

Exopolysaccharides (EPS) produced by three Lactic Acid Bacteria strains,Lactococcus lactisSLT10,Lactobacillus plantarumC7, andLeuconostoc mesenteroidesB3, were isolated using two methods: ethanol precipitation (EPS-ETOH) and ultrafiltration (EPS-UF) through a 10 KDa cut-off membrane. EPS recovery by ultrafiltration was higher than ethanol precipitation forLactococcus lactisSLT10 andLactobacillus plantarumC7. However, it was similar with both methods forLeuconostoc mesenteroidesB3. The monomer composition of the EPS fractions revealed differences in structures and molar ratios between the two studied methods. EPS isolated fromLactococcus lactisSLT10 are composed of glucose and mannose for EPS-ETOH against glucose, mannose, and rhamnose for EPS-UF. EPS extracted fromLactobacillus plantarumC7 andLeuconostoc mesenteroidesB3 showed similar composition (glucose and mannose) but different molar ratios. The molecular weights of the different EPS fractions ranged from 11.6±1.83 to 62.4±2.94 kDa. Molecular weights of EPS-ETOH fractions were higher than those of EPS-UF fractions. Fourier transform infrared (FTIR) analysis revealed a similarity in the distribution of the functional groups (O-H, C-H, C=O, -COO, and C-O-C) between the EPS isolated from the three strains.

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