scholarly journals Characterization of a Feruloyl Esterase from Lactobacillus plantarum

2013 ◽  
Vol 79 (17) ◽  
pp. 5130-5136 ◽  
Author(s):  
María Esteban-Torres ◽  
Inés Reverón ◽  
José Miguel Mancheño ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Bacterial Species ◽  
Hydrolytic Activity ◽  
Feruloyl Esterase ◽  
Gene Encoding ◽  
Content Type ◽  
Cell Extracts ◽  
Methyl Ferulate ◽  
Methyl Caffeate

ABSTRACTLactobacillus plantarumis frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant.L. plantarumWCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methylp-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methylp-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present amongL. plantarumstrains. The present study constitutes the description of feruloyl esterase activity inL. plantarumand provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species.

scholarly journals A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

2015 ◽  
Vol 81 (9) ◽  
pp. 3235-3242 ◽  
Author(s):  
María Esteban-Torres ◽  
José María Landete ◽  
Inés Reverón ◽  
Laura Santamaría ◽  
Blanca de las Rivas ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Bacterial Species ◽  
Feruloyl Esterase ◽  
Plant Origin ◽  
Methyl Gallate ◽  
Great Flexibility ◽  
Content Type ◽  
Phenolic Esters ◽  
Methyl Ferulate ◽  
Methyl Caffeate

ABSTRACTLactobacillus plantarumis the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant.L. plantarumstrains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28L. plantarumstrains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed theest_1092gene. When theest_1092gene was introduced intoL. plantarumWCFS1 orL. lactisMG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on theL. plantarumstrains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression ofest_1092was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on someL. plantarumstrains provides them with additional advantages to survive and grow on plant environments.

scholarly journals Characterization of Two Virulent Phages of Lactobacillus plantarum

2012 ◽  
Vol 78 (24) ◽  
pp. 8719-8734 ◽  
Author(s):  
Mariángeles Briggiler Marcó ◽  
Josiane E. Garneau ◽  
Denise Tremblay ◽  
Andrea Quiberoni ◽  
Sylvain Moineau
Keyword(s):  
Lactobacillus Plantarum ◽  
Corn Silage ◽  
Open Reading Frames ◽  
High Identity ◽  
Content Type ◽  
Defense Systems ◽  
Double Stranded Dna ◽  
Type Phage ◽  
Reading Frames

ABSTRACTWe characterized twoLactobacillus plantarumvirulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eightL. plantarumstrains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least twoL. plantarumstrains, LMG9211 and WCSF1. The linear double-stranded DNA genome of thepac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that ofPediococcus damnosusphage clP1 and 77% identity with that ofL. plantarumphage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of thecos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those ofBacillusandLactobacillusstrains as well as phages. Some phage B2 genes were similar to ORFs fromL. plantarumphage LP65 of theMyoviridaefamily. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.

scholarly journals Modulating Pathogenesis with Mobile-CRISPRi

2019 ◽  
Vol 201 (22) ◽  
Author(s):  
Jiuxin Qu ◽  
Neha K. Prasad ◽  
Michelle A. Yu ◽  
Shuyan Chen ◽  
Amy Lyden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Effector Proteins ◽  
Infection Model ◽  
Secretion Systems ◽  
Bacterial Genomes ◽  
Gene Encoding ◽  
Content Type ◽  
Animal Infection

ABSTRACT Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models. IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

scholarly journals The Formation of Streptococcus mutans Persisters Induced by the Quorum-Sensing Peptide Pheromone Is Affected by the LexA Regulator

2015 ◽  
Vol 197 (6) ◽  
pp. 1083-1094 ◽  
Author(s):  
Vincent Leung ◽  
Dragana Ajdic ◽  
Stephanie Koyanagi ◽  
Céline M. Lévesque
Keyword(s):  
Quorum Sensing ◽  
Streptococcus Mutans ◽  
Bacterial Species ◽  
Regulatory System ◽  
Bacterial Survival ◽  
Chronic Infections ◽  
Persister Cells ◽  
Gene Encoding ◽  
Content Type ◽  
Peptide Pheromone

The presence of multidrug-tolerant persister cells within microbial populations has been implicated in the resiliency of bacterial survival against antibiotic treatments and is a major contributing factor in chronic infections. The mechanisms by which these phenotypic variants are formed have been linked to stress response pathways in various bacterial species, but many of these mechanisms remain unclear. We have previously shown that in the cariogenic organismStreptococcus mutans, the quorum-sensing peptide CSP (competence-stimulating peptide) pheromone was a stress-inducible alarmone that triggered an increased formation of multidrug-tolerant persisters. In this study, we characterized SMU.2027, a CSP-inducible gene encoding a LexA ortholog. We showed that in addition to exogenous CSP exposure, stressors, including heat shock, oxidative stress, and ofloxacin antibiotic, were capable of triggering expression oflexAin an autoregulatory manner akin to that of LexA-like transcriptional regulators. We demonstrated the role of LexA and its importance in regulating tolerance toward DNA damage in a noncanonical SOS mechanism. We showed its involvement and regulatory role in the formation of persisters induced by the CSP-ComDE quorum-sensing regulatory system. We further identified key genes involved in sugar and amino acid metabolism, the clustered regularly interspaced short palindromic repeat (CRISPR) system, and autolysin from transcriptomic analyses that contribute to the formation of quorum-sensing-induced persister cells.

scholarly journals Tannin Degradation by a Novel Tannase Enzyme Present in Some Lactobacillus plantarum Strains

2014 ◽  
Vol 80 (10) ◽  
pp. 2991-2997 ◽  
Author(s):  
Natalia Jiménez ◽  
María Esteban-Torres ◽  
José Miguel Mancheño ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Tannic Acid ◽  
Plant Material ◽  
Aliphatic Alcohol ◽  
Methyl Gallate ◽  
Content Type ◽  
Sequence Identity ◽  
Tannin Degradation ◽  
Identification And Characterization

ABSTRACTLactobacillus plantarumis frequently isolated from the fermentation of plant material where tannins are abundant.L. plantarumstrains possess tannase activity to degrade plant tannins. AnL. plantarumtannase (TanBLp, formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanALp). While all 29L. plantarumstrains analyzed in the study possess thetanBLpgene, the genetanALpwas present in only four strains. Upon methyl gallate exposure, the expression oftanBLpwas induced, whereastanALpexpression was not affected. TanALpshowed only 27% sequence identity to TanBLp, but the residues involved in tannase activity are conserved. Optimum activity for TanALpwas observed at 30°C and pH 6 in the presence of Ca2+ions. TanALpwas able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanALpwas able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanALptannase in someL. plantarumstrains provides them an advantage for the initial degradation of complex tannins present in plant environments.

Characterization of Muscle Sarcoplasmic and Myofibrillar Protein Hydrolysis Caused by Lactobacillus plantarum

1999 ◽  
Vol 65 (8) ◽  
pp. 3540-3546 ◽  
Author(s):  
Silvina Fadda ◽  
Yolanda Sanz ◽  
Graciela Vignolo ◽  
M.-Concepción Aristoy ◽  
Guillermo Oliver ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Myofibrillar Protein ◽  
Myofibrillar Proteins ◽  
Muscle Proteins ◽  
Whole Cells ◽  
Sarcoplasmic Proteins ◽  
Cell Extracts ◽  
Hydrophobic Nature ◽  
Hydrolysis Of

ABSTRACT Strains of Lactobacillus plantarum originally isolated from sausages were screened for proteinase and aminopeptidase activities toward synthetic substrates; on the basis of that screening,L. plantarum CRL 681 was selected for further assays on muscle proteins. The activities of whole cells, cell extracts (CE), and a combination of both on sarcoplasmic and myofibrillar protein extracts were determined by protein, peptide, and free-amino-acid analyses. Proteinase from whole cells initiated the hydrolysis of sarcoplasmic proteins. The addition of CE intensified the proteolysis. Whole cells generated hydrophilic peptides from both sarcoplasmic and myofibrillar proteins. Other peptides of a hydrophobic nature resulted from the combination of whole cells and CE. The action of both enzymatic sources on myofibrillar proteins caused maximal increases in lysine, arginine, and leucine, while the action of those on sarcoplasmic proteins mainly released alanine. In general, pronounced hydrolysis of muscle proteins required enzyme activities from whole cells in addition to those supplied by CE.

scholarly journals Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

2011 ◽  
Vol 77 (20) ◽  
pp. 7104-7112 ◽  
Author(s):  
Maria Karczmarczyk ◽  
Yvonne Abbott ◽  
Ciara Walsh ◽  
Nola Leonard ◽  
Séamus Fanning
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Gene Array ◽  
Multidrug Resistant ◽  
Genetic Determinants ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Class 1

ABSTRACTIn this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection ofEscherichia coliisolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1,dfrA1-aadA1,dfrA17-aadA5,dfrA12-orfF-aadA2,blaOXA-30-aadA1,aacC1-orf1-orf2-aadA1,dfr7). Class 2 integrons (13.5%) contained thedfrA1-sat1-aadA1gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected includedblaTEM,cat,floR,aadB,aphA1,strA-strB,sul2, andtet(B), respectively. TheblaCTX-M-2gene, encoding an extended-spectrum β-lactamase (ESβL), andblaCMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensalE. coliisolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, theblaCTX-M-2gene has not previously been reported in Ireland.

scholarly journals Genomic Characterization of VIM Metallo-β-Lactamase-Producing Alcaligenes faecalis from Gaza, Palestine

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Nahed Al Laham ◽  
Kalyan D. Chavda ◽  
Astrid V. Cienfuegos-Gallet ◽  
Barry N. Kreiswirth ◽  
Liang Chen
Keyword(s):  
Bacterial Species ◽  
Alcaligenes Faecalis ◽  
Gram Negative Bacteria ◽  
Sequencing Analysis ◽  
Large Hospital ◽  
Content Type ◽  
Next Generation Sequencing Analysis ◽  
Genomic Characterization ◽  
Generation Sequencing

ABSTRACT Carbapenemase-producing Gram-negative bacteria (CP-GNB) have increasingly spread worldwide, and different families of carbapenemases have been identified in various bacterial species. Here, we report the identification of five VIM metallo-β-lactamase-producing Alcaligenes faecalis isolates associated with a small outbreak in a large hospital in Gaza, Palestine. Next-generation sequencing analysis showed bla VIM-2 is harbored by a chromosomal genomic island among three strains, while bla VIM-4 is carried by a novel plasmid in two strains.

scholarly journals Genetic and Biochemical Characterization of FRI-1, a Carbapenem-Hydrolyzing Class A β-Lactamase from Enterobacter cloacae

2015 ◽  
Vol 59 (12) ◽  
pp. 7420-7425 ◽  
Author(s):  
Laurent Dortet ◽  
Laurent Poirel ◽  
Samia Abbas ◽  
Saoussen Oueslati ◽  
Patrice Nordmann
Keyword(s):  
Biochemical Characterization ◽  
Enterobacter Cloacae ◽  
Conjugative Plasmid ◽  
Lower Sensitivity ◽  
Gene Encoding ◽  
Content Type ◽  
Class A ◽  
Encoding Gene ◽  
Lactamase Gene

ABSTRACTAnEnterobacter cloacaeisolate was recovered from a rectal swab from a patient hospitalized in France with previous travel to Switzerland. It was resistant to penicillins, narrow- and broad-spectrum cephalosporins, aztreonam, and carbapenems but remained susceptible to expanded-spectrum cephalosporins. Whereas PCR-based identification of the most common carbapenemase genes failed, the biochemical Carba NP test II identified an Ambler class A carbapenemase. Cloning experiments followed by sequencing identified a gene encoding a totally novel class A carbapenemase, FRI-1, sharing 51 to 55% amino acid sequence identity with the closest carbapenemase sequences. However, it shared conserved residues as a source of carbapenemase activity. Purified β-lactamase FRI-1 hydrolyzed penicillins, aztreonam, and carbapenems but spared expanded-spectrum cephalosporins. The 50% inhibitory concentrations (IC50s) of clavulanic acid and tazobactam were 10-fold higher than those found forKlebsiella pneumoniaecarbapenemase (KPC), IMI, and SME, leading to lower sensitivity of FRI-1 activity to β-lactamase inhibitors. TheblaFRI-1gene was located on a ca. 110-kb untypeable, transferable, and non-self-conjugative plasmid. A putative LysR family regulator-encoding gene at the 5′ end of the β-lactamase gene was identified, leading to inducible expression of theblaFRI-1gene.

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

2015 ◽  
Vol 81 (17) ◽  
pp. 5993-6002 ◽  
Author(s):  
Winschau F. van Zyl ◽  
Shelly M. Deane ◽  
Leon M. T. Dicks
Keyword(s):  
Lactobacillus Plantarum ◽  
Fluorescent Protein ◽  
Intestinal Colonization ◽  
Red Fluorescent Protein ◽  
Gene Encoding ◽  
Content Type ◽  
Enterococcus Mundtii ◽  
Probiotic Strains ◽  
Reporter Systems

ABSTRACTLactic 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 thein vivotagging of the probiotic strainsEnterococcus mundtiiST4SA andLactobacillus plantarum423. ThemCherrygene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome ofL. plantarum423 by homologous recombination. In the case ofE. mundtiiST4SA, themCherrygene was cloned into the pGKV223D LAB/Escherichia coliexpression vector. Expression of themCherrygene 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.

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