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 Substrate Inhibition of VanA by d-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

2016 ◽  
Vol 60 (8) ◽  
pp. 4930-4939 ◽  
Author(s):  
Lizah T. van der Aart ◽  
Nicole Lemmens ◽  
Willem J. van Wamel ◽  
Gilles P. van Wezel
Keyword(s):  
Cell Wall ◽  
Streptomyces Coelicolor ◽  
Substrate Inhibition ◽  
Model Organism ◽  
Vancomycin Resistance ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
Lipid Ii ◽  
Vancomycin Resistant

ABSTRACTThe increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to thed-Ala–d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variantd-Ala–d-Lac, which is produced by VanA. Here we show that exogenousd-Ala competes withd-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of thed-Ala–d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations ofd-Ala led to the production of a significant amount of wild-type cell wall precursors, whilevanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organismStreptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates ofEnterococcus faecium(VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity ofS. coelicolorcells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance.

scholarly journals CRISPR/Cas9-Assisted Seamless Genome Editing in Lactobacillus plantarum and Its Application in N-Acetylglucosamine Production

2019 ◽  
Vol 85 (21) ◽  
Author(s):  
Ding Zhou ◽  
Zhennan Jiang ◽  
Qingxiao Pang ◽  
Yuan Zhu ◽  
Qian Wang ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Genome Editing ◽  
Lactobacillus Plantarum ◽  
Strain Engineering ◽  
Single Stranded Dna ◽  
Content Type ◽  
Recombination Efficiency ◽  
Double Stranded Dna ◽  
Exogenous Genes

ABSTRACT Lactobacillus plantarum is a potential starter and health-promoting probiotic bacterium. Effective, precise, and diverse genome editing of Lactobacillus plantarum without introducing exogenous genes or plasmids is of great importance. In this study, CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering was established in L. plantarum WCFS1 to seamlessly edit the genome, including gene knockouts, insertions, and point mutations. To optimize our editing method, phosphorothioate modification was used to improve the dsDNA insertion, and adenine-specific methyltransferase was used to improve the ssDNA recombination efficiency. These strategies were applied to engineer L. plantarum WCFS1 toward producing N-acetylglucosamine (GlcNAc). nagB was truncated to eliminate the reverse reaction of fructose-6-phosphate (F6P) to glucosamine 6-phosphate (GlcN-6P). Riboswitch replacement and point mutation in glmS1 were introduced to relieve feedback repression. The resulting strain produced 797.3 mg/liter GlcNAc without introducing exogenous genes or plasmids. This strategy may contribute to the available methods for precise and diverse genetic engineering in lactic acid bacteria and boost strain engineering for more applications. IMPORTANCE CRISPR/Cas9-assisted recombineering is restricted in lactic acid bacteria because of the lack of available antibiotics and vectors. In this study, a seamless genome editing method was carried out in Lactobacillus plantarum using CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering, and recombination efficiency was effectively improved by endogenous adenine-specific methyltransferase overexpression. L. plantarum WCFS1 produced 797.3 mg/liter N-acetylglucosamine (GlcNAc) through reinforcement of the GlcNAc pathway, without introducing exogenous genes or plasmids. This seamless editing strategy, combined with the potential exogenous GlcNAc-producing pathway, makes this strain an attractive candidate for industrial use in the future.

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 Core Fluxome and Metafluxome of Lactic Acid Bacteria under Simulated Cocoa Pulp Fermentation Conditions

2013 ◽  
Vol 79 (18) ◽  
pp. 5670-5681 ◽  
Author(s):  
Philipp Adler ◽  
Christoph Josef Bolten ◽  
Katrin Dohnt ◽  
Carl Erik Hansen ◽  
Christoph Wittmann
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Starter Cultures ◽  
Superior Performance ◽  
Gas Chromatography Mass Spectrometry ◽  
Glycolytic Flux ◽  
Content Type ◽  
Phosphoketolase Pathway ◽  
Labeling Approach ◽  
Cocoa Fermentation

ABSTRACTIn the present work, simulated cocoa fermentation was investigated at the level of metabolic pathway fluxes (fluxome) of lactic acid bacteria (LAB), which are typically found in the microbial consortium known to convert nutrients from the cocoa pulp into organic acids. A comprehensive13C labeling approach allowed to quantify carbon fluxes during simulated cocoa fermentation by (i) parallel13C studies with [13C6]glucose, [1,2-13C2]glucose, and [13C6]fructose, respectively, (ii) gas chromatography-mass spectrometry (GC/MS) analysis of secreted acetate and lactate, (iii) stoichiometric profiling, and (iv) isotopomer modeling for flux calculation. The study of several strains ofL. fermentumandL. plantarumrevealed major differences in their fluxes. TheL. fermentumstrains channeled only a small amount (4 to 6%) of fructose into central metabolism, i.e., the phosphoketolase pathway, whereas onlyL. fermentumNCC 575 used fructose to form mannitol. In contrast,L. plantarumstrains exhibited a high glycolytic flux. All strains differed in acetate flux, which originated from fractions of citrate (25 to 80%) and corresponding amounts of glucose and fructose. Subsequent, metafluxome studies with consortia of differentL. fermentumandL. plantarumstrains indicated a dominant (96%) contribution ofL. fermentumNCC 575 to the overall flux in the microbial community, a scenario that was not observed for the other strains. This highlights the idea that individual LAB strains vary in their metabolic contribution to the overall fermentation process and opens up new routes toward streamlined starter cultures.L. fermentumNCC 575 might be one candidate due to its superior performance in flux activity.

Cell-free supernatants produced by lactic acid bacteria reduce Salmonella population in vitro

Microbiology ◽  
2021 ◽  
Vol 167 (11) ◽  
Author(s):  
Alberto Gonçalves Evangelista ◽  
Jessica Audrey Feijó Corrêa ◽  
João Vitor Garcia dos Santos ◽  
Eduardo Henrique Custódio Matté ◽  
Mônica Moura Milek ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Lactic Acid ◽  
Antimicrobial Resistance ◽  
Lactic Acid Bacteria ◽  
Type Species ◽  
Feed Additives ◽  
Poultry Feed ◽  
Content Type ◽  
Link Type

The genus Salmonella is closely associated with foodborne outbreaks and animal diseases, and reports of antimicrobial resistance in Salmonella species are frequent. Several alternatives have been developed to control this pathogen, such as cell-free supernatants (CFS). Our objective here was to evaluate the use of lactic acid bacteria (LAB) CFS against Salmonella in vitro. Seventeen strains of LAB were used to produce CFS, and their antimicrobial activity was screened towards six strains of Salmonella . In addition, CFS were also pH-neutralized and/or boiled. Those with the best results were lyophilized. MICs of lyophilized CFS were 11.25–22.5 g l–1. Freeze-dried CFS were also used to supplement swine and poultry feed (11.25 g kg–1) and in vitro simulated digestion of both species was performed, with Salmonella contamination of 5×106 and 2×105 c.f.u. g−1 of swine and poultry feed, respectively. In the antimicrobial screening, all acidic CFS were able to inhibit the growth of Salmonella . After pH neutralization, Lactobacillus acidophilus Llorente, Limosilactobacillus fermentum CCT 1629, Lactiplantibacillus plantarum PUCPR44, Limosilactobacillus reuteri BioGaia, Lacticaseibacillus rhamnosus ATCC 7469 and Pediococcus pentosaceus UM116 CFS were the only strains that partially maintained their antimicrobial activity and, therefore, were chosen for lyophilization. In the simulated swine digestion, Salmonella counts were reduced ≥1.78 log c.f.u. g–1 in the digesta containing either of the CFS. In the chicken simulation, a significant reduction was obtained with all CFS used (average reduction of 0.59±0.01 log c.f.u. ml–1). In general, the lyophilized CFS of L. fermentum CCT 1629, L. rhamnosus ATCC 7469 and L. acidophilus Llorente presented better antimicrobial activity. In conclusion, CFS show potential as feed additives to control Salmonella in animal production and may be an alternative to the use of antibiotics, minimizing problems related to antimicrobial resistance.

scholarly journals Complete Genome Sequence of Lactobacillus reuteri Byun-re-01, Isolated from Mouse Small Intestine

2018 ◽  
Vol 7 (17) ◽  
Author(s):  
Dongjun Kim ◽  
Mun-ju Cho ◽  
Seungchan Cho ◽  
Yongjun Lee ◽  
Sung June Byun ◽  
...  
Keyword(s):  
Small Intestine ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Lactobacillus Reuteri ◽  
Probiotic Bacteria ◽  
Content Type ◽  
Mouse Small Intestine

Lactic acid bacteria (LAB) are generally recognized as safe (GRAS) and serve as probiotic bacteria when consumed in adequate amounts. Here, we report the complete genome sequence of Lactobacillus reuteri Byun-re-01, isolated from mouse small intestine.

scholarly journals Capacity of Human Nisin- and Pediocin-Producing Lactic Acid Bacteria To Reduce Intestinal Colonization by Vancomycin-Resistant Enterococci

2008 ◽  
Vol 74 (7) ◽  
pp. 1997-2003 ◽  
Author(s):  
Mathieu Millette ◽  
Gilbert Cornut ◽  
Claude Dupont ◽  
François Shareck ◽  
Denis Archambault ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
In Vivo Study ◽  
Control Group ◽  
Intestinal Colonization ◽  
Strong Activity ◽  
Vancomycin Resistant Enterococci ◽  
Significant Difference ◽  
Vancomycin Resistant

ABSTRACT This study demonstrated the capacity of bacteriocin-producing lactic acid bacteria (LAB) to reduce intestinal colonization by vancomycin-resistant enterococci (VRE) in a mouse model. Lactococcus lactis MM19 and Pediococcus acidilactici MM33 are bacteriocin producers isolated from human feces. The bacteriocin secreted by P. acidilactici is identical to pediocin PA-1/AcH, while PCR analysis demonstrated that L. lactis harbors the nisin Z gene. LAB were acid and bile tolerant when assayed under simulated gastrointestinal conditions. A well diffusion assay using supernatants from LAB demonstrated strong activity against a clinical isolate of VRE. A first in vivo study was done using C57BL/6 mice that received daily intragastric doses of L. lactis MM19, P. acidilactici MM33, P. acidilactici MM33A (a pediocin mutant that had lost its ability to produce pediocin), or phosphate-buffered saline (PBS) for 18 days. This study showed that L. lactis and P. acidilactici MM33A increased the concentrations of total LAB and anaerobes while P. acidilactici MM33 decreased the Enterobacteriaceae populations. A second in vivo study was done using VRE-colonized mice that received the same inocula as those in the previous study for 16 days. In L. lactis-fed mice, fecal VRE levels 1.73 and 2.50 log10 CFU/g lower than those in the PBS group were observed at 1 and 3 days postinfection. In the P. acidilactici MM33-fed mice, no reduction was observed at 1 day postinfection but a reduction of 1.85 log10 CFU/g was measured at 3 days postinfection. Levels of VRE in both groups of mice treated with bacteriocin-producing LAB were undetectable at 6 days postinfection. No significant difference in mice fed the pediocin-negative strain compared to the control group was observed. This is the first demonstration that human L. lactis and P. acidilactici nisin- and pediocin-producing strains can reduce VRE intestinal colonization.

scholarly journals Intracellular Free Iron and Its Potential Role in Ultrahigh-Pressure-Induced Inactivation of Escherichia coli

2012 ◽  
Vol 79 (2) ◽  
pp. 722-724 ◽  
Author(s):  
Yuan Yan ◽  
Joy G. Waite-Cusic ◽  
Periannan Kuppusamy ◽  
Ahmed E. Yousef
Keyword(s):  
Escherichia Coli ◽  
Iron Chelator ◽  
Ultrahigh Pressure ◽  
Dependent Manner ◽  
Paramagnetic Resonance ◽  
Free Iron ◽  
Content Type ◽  
E Coli ◽  
Electron Paramagnetic ◽  
Dose Dependent

ABSTRACTIntracellular free iron ofEscherichia coliwas determined by whole-cell electron paramagnetic resonance spectrometry. Ultrahigh pressure (UHP) increased both intracellular free iron and cell lethality in a pressure-dose-dependent manner. The iron chelator 2,2′-dipyridyl protected cells against UHP treatments. A mutation that produced iron overload conditions sensitizedE. colito UHP treatment.

scholarly journals Ethylphenol Formation byLactobacillus plantarum: Identification of the Enzyme Involved in the Reduction of Vinylphenols

2018 ◽  
Vol 84 (17) ◽  
Author(s):  
Laura Santamaría ◽  
Inés Reverón ◽  
Félix López de Felipe ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Bacterial Species ◽  
Alcoholic Beverages ◽  
Genetic Identification ◽  
Detection Methods ◽  
Fermented Foods ◽  
Content Type ◽  
Volatile Phenol ◽  
Off Flavors

ABSTRACTEthylphenols are strong odorants produced by microbial activity that are described as off flavors in several foods.Lactobacillus plantarumis a lactic acid bacterial species able to produce ethylphenols by the reduction of vinylphenols during the metabolism of hydroxycinnamic acids. However, the reductase involved has not been yet uncovered. In this study, the involvement in vinylphenol reduction of a gene encoding a putative reductase (lp_3125) was confirmed by the absence of reduction activity in the Δlp_3125knockout mutant. The protein encoded bylp_3125, VprA, was recombinantly produced inEscherichia coli. VprA was assayed against vinylphenols (4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol), and all were reduced to their corresponding ethylphenols (4-ethylphenol, 4-ethylcatechol, and 4-ethylguaiacol). PCR and high-performance liquid chromatography (HPLC) detection methods revealed that the VprA reductase is not widely distributed among the lactic acid bacteria studied and that only the bacteria possessing thevprAgene were able to produce ethylphenol from vinylphenol. However, all the species belonging to theL. plantarumgroup were ethylphenol producers. The identification of theL. plantarumVprA protein involved in hydroxycinnamate degradation completes the route of degradation of these compounds in lactic acid bacteria.IMPORTANCEThe presence of volatile phenols is considered a major organoleptic defect of several fermented alcoholic beverages. The biosynthesis of these compounds has been mainly associated withBrettanomyces/Dekkerayeasts. However, the potential importance of lactic acid bacteria in volatile phenol spoilage is emphasized by reports describing a faster ethylphenol production by these bacteria than by yeasts. The genetic identification of the bacterial vinylphenol reductase involved in volatile phenol production provides new insights into the role of lactic acid bacteria in the production of these off flavors. The development of a molecular method for the detection of ethylphenol-producing bacteria could be helpful to design strategies to reduce the bacterial production of vinylphenols in fermented foods.

scholarly journals Draft Genome Sequence of Lactococcus sp. Strain NtB2 (JCM 32569), Isolated from the Gut of the Higher Termite Nasutitermes takasagoensis

2018 ◽  
Vol 6 (24) ◽  
Author(s):  
Satoko Noda ◽  
Chihiro Aihara ◽  
Masahiro Yuki ◽  
Moriya Ohkuma
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Genome Sequence ◽  
Draft Genome ◽  
Draft Genome Sequence ◽  
Content Type ◽  
Termite Gut

ABSTRACT Lactic acid bacteria are widely distributed in the termite gut. Here, we report the draft genome sequence of Lactococcus sp. strain NtB2, which was isolated from the gut of a wood-feeding higher termite.

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