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1

The Utilization of Potassium by Bact. lactis aerogenes  

Microsoft Academic Search

As the potassium-ion concentration of synthetic media is decreased the total population (ns) of Bact. lactis aerogenes which they support tends towards zero. Added potassium gives nearly linear increases in ns. The use of the radioactive isotope shows that the cells take up nearly all the potassium from the medium over wide ranges of concentration. Sodium cannot replace potassium, and

A. A. Eddy; Cyril Hinshelwood

1950-01-01

2

DU COLOSTRUM 38 fois les bact. Gram -  

E-print Network

DU COLOSTRUM 38 fois les bact. Gram - 33 fois les bact. Gram + 19 fois les bact. Gram + et Gram - à- taté : 36 fois les bact. Gram - 39 fois les bact. Gram + 18 fois les bact. Gram - et Gram + 3 fois

Paris-Sud XI, Université de

3

Serine utilization by Klebsiella aerogenes.  

PubMed Central

Klebsiella aerogenes was found to contain a specific L-serine dehydrase that was induced by threonine, glycine or leucine, but not by its substrate. Cellular concentrations were sensitive to carbon rather than nitrogen sources in the growth medium. A nonspecific isoleucine-sensitive L-threonine dehydrase supplemented the specific L-serine dehydrase activity. K. aerogenes also contains a leucine-inducible L-threonine dehydrogenase which probably initiated a threonine-utilization pathway in which the serine-specific dehydrate participated. Strains that were altered in their ability to metabolize serine differed in either L-serine dehydrase or L-threonine dehydrase activity. Thus, K. aerogenes growing on L-serine as a sole nitrogen source relies upon two enzymes that metabolize the amino acid as subsidiary functions. PMID:6783624

Vining, L C; Magasanik, B

1981-01-01

4

Amino acid utilization by Aerobacter aerogenes and Escherichia coli  

E-print Network

A considerable amount of work has been done on the growth of A. aerogenes and E. coli in synthetic media, but little work has been undertaken on the utilization by these organisms of amino acids as comparative sources of ...

Herrera, Rodolfo Eduardo

1938-01-01

5

Protocatechuate is not metabolized via catechol in Enterobacter aerogenes.  

PubMed Central

Protocatechuate is generally metabolized in bacteria by direct oxygenative cleavage to produce beta-carboxymuconate. An exception to this pattern has been suggested by reports that protocatechuate might be metabolized by nonoxidative decarboxylation to catechol in Enterobacter aerogenes. In the present investigation, analysis of mutant strains indicated that this proposed pathway did not make a significant contribution to protocatechuate metabolism in E. aerogenes because mutations blocking catechol metabolism did not impair protocatechuate utilization. In addition, all the enzymes required for the oxygenative cleavage of protocatechuate and its further metabolism were induced in E. aerogenes during protocatechuate metabolism, and mutations inactivating this oxygenative pathway prevented protocatechuate degradation. The strains of E. aerogenes examined exhibited broad specificities of inductive control over genes associated with protocatechuate and catechol metabolism; it appears that a number of metabolites may trigger the expression of these genes. PMID:3680179

Doten, R C; Ornston, L N

1987-01-01

6

On the amine oxidases of Klebsiella aerogenes strain W70.  

PubMed

Klebsiella aerogenes W70 was reported previously to produce a membrane-associated tyramine oxidase (TynA) that did not act on 2-phenylethylamine. Subsequently, a gene cloned from K. aerogenes W70 produced a soluble amine oxidase (MaoA) that acted readily on 2-phenylethylamine and tyramine. This enzyme appeared to be equivalent to a 2-phenylethylamine oxidase of Escherichia coli K-12 (MaoA) but was assumed to be the originally described K. aerogenes W70 tyramine oxidase (TynA). However, as described here, whole cells and cell-free extracts of K. aerogenes W70 showed only the tyramine oxidase (TynA) that is inactive against 2-phenylethylamine and not the maoA gene product. It seems that the organism has two amine oxidase genes, tynA and maoA, but only tynA is expressed. Hence, data relating to the expression of the K. aerogenes W70 tynA gene cannot be assumed to apply to the maoA gene of E. coli K-12 because they encode different enzymes. PMID:8997710

Cooper, R A

1997-01-01

7

Natural immune systems protect animals from dangerous foreign pathogens, including bacte-  

E-print Network

Natural immune systems protect animals from dangerous foreign pathogens, including bacte- ria computer immune systems with some of the important properties of natural immune systems, including are less well known. The immune system provides a persuasive example of how they might be implemented

Garlan, David

8

Magnesium-limited growth of Aerobacter aerogenes in a chemostat  

Microsoft Academic Search

SUMMARY The influence of Mg2+-lirnitationy compared with carbon-limitation, on bacterial concentration, and on protein, carbohydrate, RNA and DNA contents of Aerobacter aerogenes cultures (grown in the chemostat at several dilution rates) was determined. In both types of culture the bacterial protein, carbohydrate and DNA contents varied slightly, and the RNA content grossly, with changes in dilution rate. Bacterial yield varied

D. W. TEMPEST; J. R. HUNTER; J. SYKES

1965-01-01

9

Biological Conversion of Glycerol to Ethanol by Enterobacter aerogenes  

NASA Astrophysics Data System (ADS)

In a search to turn the economically and environmentally non-valuable "waste" streams of biodiesel production into a profitable byproduct, a mutant strain of Enterobacter aerogenes ATCC 13048 was developed by six-tube subculturing technique. This technique is based on the principle of adaptive evolution, and involved subculturing the bacterium in a tryptic soy broth without dextrose (TSB) containing specific glycerol and ethanol concentration for six consecutive times. Then, the six consecutive subculturing was repeated in a fresh TSB of higher glycerol and ethanol concentrations. A new mutant strain, E. aerogenes S012, which could withstand a combination of 200 g/l glycerol and 30 g/l ethanol concentrations, was developed. The wild and mutant strains were used for the fermentation of pure (P-) and recovered (R-) glycerol. Taguchi and full factorial methods of design of experiments were used to screen and optimize the important process factors that influence the microbial production of ethanol. A statistically sound regression model was used to establish the mathematical relationship between the process variables and ethanol production. Temperature of 38C, agitation speed of 200 rpm, pH of 6.3-6.6, and microaerobic condition were the optimum process conditions. Different pretreatment methods to recover glycerol from the crude glycerol and the subsequent fermentation method showed that direct acidification using 85% H3PO4 was the best. The R-glycerol contained 51% pure glycerol and 21% methanol. The wild strain, E. aerogenes ATCC 13048, produced only 12 g/l and 12.8 g/l ethanol from 20 g/l P- and R-glycerol respectively, and could not utilize higher glycerol concentrations. The mutant, E. aerogenes S012, produced ethanol amount and yield of 43 g/l and 1.12 mol/mol-glycerol from P-glycerol, respectively within 96 h. It also produced ethanol amount and yield of 26.8 g/l and 1.07 mol/mol-glycerol, respectively, from R-glycerol within the same duration. In a fermentation to estimate hydrogen production using a respirometer, the hydrogen yield and volumetric rate of 1.06 mol/mol-glycerol and 217 ml/l/h, respectively were obtained from 6% P-glycerol in 72 h by E. aerogenes S012. The result was higher from R-glycerol, which produced hydrogen yield and productivity of 1.83 mol/mol-glycerol and 326 ml/l/h, respectively.

Nwachukwu, Raymond E. S.

10

Lactococcus lactis subsp. lactis infection in waterfowl: first confirmation in animals.  

PubMed Central

We report the first description, confirmed by bacteriologic and molecular (polymerase chain reaction and pulsed-field gel electrophoresis) analysis, of an infection in animals caused by Lactococcus lactis subsp. lactis, affecting waterfowl. PMID:11747704

Goyache, J.; Vela, A. I.; Gibello, A.; Blanco, M. M.; Briones, V.; Gonzlez, S.; Tllez, S.; Ballesteros, C.; Domnguez, L.; Fernndez-Garayzbal, J. F.

2001-01-01

11

Exploring the Genome of Cheese Starter Lactic Acid Bacterium Lactococcus lactis subsp. lactis CECT 4433  

PubMed Central

Here, we present the draft genome sequences of Lactococcus lactis subsp. lactis CECT 4433, a cheese fermentation starter strain. The genome provides further insight into the genomic plasticity, biocomplexity (including gene strain specifics), and evolution of these genera. PMID:25395632

Tschoeke, Diogo Antonio; Moreira, Ana Paula B.; Chimetto Tonon, Luciane A.; de Mesquita, Milene Miranda A.; Gregoracci, Gustavo B.; Gomez-Gil, Bruno; Valle, Rogrio; Thompson, Cristiane C.

2014-01-01

12

Genetic control of arylsulfatase synthesis in Klebsiella aerogenes.  

PubMed

It was shown that at least four genes are specifically responsible for arylsulfatase synthesis in Klebsiella aerogenes. Mutations at chromosome site atsA result in enzymatically inactive arylsulfatase. Mutants showing constitutive synthesis of arylsulfatase (atsR) were isolated by using inorganic sulfate or cysteine as the sulfur source. Another mutation in which repression of arylsulfatase by inorganic sulfate or cysteine could not be relieved by tyramine was determined by genetic analysis to be on the tyramine oxidase gene (tyn). This site was distinguished from the atsC mutation site, which is probably concerned with the action or synthesis of corepressors of arylsulfatase synthesis. Genetic analysis with transducing phage PW52 showed that the order of mutation sites was atsC-atsR-atsA-tynA-tynB. On the basis of these results and previous physiological findings, we propose a new model for regulation of arylsulfatase synthesis. PMID:853036

Murooka, Y; Adachi, T; Okamura, H; Harada, T

1977-04-01

13

Genetic control of arylsulfatase synthesis in Klebsiella aerogenes.  

PubMed Central

It was shown that at least four genes are specifically responsible for arylsulfatase synthesis in Klebsiella aerogenes. Mutations at chromosome site atsA result in enzymatically inactive arylsulfatase. Mutants showing constitutive synthesis of arylsulfatase (atsR) were isolated by using inorganic sulfate or cysteine as the sulfur source. Another mutation in which repression of arylsulfatase by inorganic sulfate or cysteine could not be relieved by tyramine was determined by genetic analysis to be on the tyramine oxidase gene (tyn). This site was distinguished from the atsC mutation site, which is probably concerned with the action or synthesis of corepressors of arylsulfatase synthesis. Genetic analysis with transducing phage PW52 showed that the order of mutation sites was atsC-atsR-atsA-tynA-tynB. On the basis of these results and previous physiological findings, we propose a new model for regulation of arylsulfatase synthesis. PMID:853036

Murooka, Y; Adachi, T; Okamura, H; Harada, T

1977-01-01

14

The Citrate Transport System of Lactococcus lactis subsp. lactis biovar diacetylactis Is Induced by Acid Stress  

Microsoft Academic Search

Citrate transport in Lactococcus lactis subsp. lactis biovar diacetylactis is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic citP gene. We have shown previously that citP is included in the citQRP operon, which is mainly transcribed from the P1 promoter in L. lactis subsp. lactis biovar diacetylactis. Furthermore, transcription of citQRP and citrate transport are not

NIEVES GARCIA-QUINTANS; CHRISTIAN MAGNI; DIEGO DE MENDOZA; PALOMA LOPEZ

1998-01-01

15

Bioconversion of glycerol to ethanol by a mutant Enterobacter aerogenes  

PubMed Central

The main objective of this research is to develop, by adaptive evolution, mutant strains of Enterobacter aerogenes ATCC 13048 that are capable of withstanding high glycerol concentration as well as resisting ethanol-inhibition. The mutant will be used for high ethanol fermentation from glycerol feedstock. Ethanol production from pure (P-) and recovered (R-) glycerol using the stock was evaluated. A six-tube-subculture-generations method was used for developing the mutant. This involved subculturing the organism six consecutive times in tubes containing the same glycerol and ethanol concentrations at the same culture conditions. Then, the glycerol and/or ethanol concentration was increased and the six subculture generations were repeated. A strain capable of growing in 200 g/L glycerol and 30 g/L ethanol was obtained. The ability of this mutant, vis--vis the original strain, in utilizing glycerol in a high glycerol containing medium, with the concomitant ethanol yield, was assessed. Tryptic soy broth without dextrose (TSB) was used as the fermentation medium. Fermentation products were analyzed using HPLC. In a 20 g/L glycerol TSB, E. aerogenes ATCC 13048 converted 18.5 g/L P-glycerol and 17.8 g/L R-glycerol into 12 and 12.8 g/L ethanol, respectively. In a 50 g/L P-glycerol TSB, it utilized only 15.6 g/L glycerol; but the new strain used up 39 g/L, yielding 20 g/L ethanol after 120 h, an equivalence of 1.02 mol ethanol/mol-glycerol. This is the highest ethanol yield reported from glycerol bioconversion. The result of this P-glycerol fermentation can be duplicated using the R-glycerol from biodiesel production. PMID:22455837

2012-01-01

16

Bioconversion of glycerol to ethanol by a mutant Enterobacter aerogenes.  

PubMed

The main objective of this research is to develop, by adaptive evolution, mutant strains of Enterobacter aerogenes ATCC 13048 that are capable of withstanding high glycerol concentration as well as resisting ethanol-inhibition. The mutant will be used for high ethanol fermentation from glycerol feedstock. Ethanol production from pure (P-) and recovered (R-) glycerol using the stock was evaluated. A six-tube-subculture-generations method was used for developing the mutant. This involved subculturing the organism six consecutive times in tubes containing the same glycerol and ethanol concentrations at the same culture conditions. Then, the glycerol and/or ethanol concentration was increased and the six subculture generations were repeated. A strain capable of growing in 200 g/L glycerol and 30 g/L ethanol was obtained. The ability of this mutant, vis--vis the original strain, in utilizing glycerol in a high glycerol containing medium, with the concomitant ethanol yield, was assessed. Tryptic soy broth without dextrose (TSB) was used as the fermentation medium. Fermentation products were analyzed using HPLC.In a 20 g/L glycerol TSB, E. aerogenes ATCC 13048 converted 18.5 g/L P-glycerol and 17.8 g/L R-glycerol into 12 and 12.8 g/L ethanol, respectively. In a 50 g/L P-glycerol TSB, it utilized only 15.6 g/L glycerol; but the new strain used up 39 g/L, yielding 20 g/L ethanol after 120 h, an equivalence of 1.02 mol ethanol/mol-glycerol. This is the highest ethanol yield reported from glycerol bioconversion. The result of this P-glycerol fermentation can be duplicated using the R-glycerol from biodiesel production. PMID:22455837

Nwachukwu, Res; Shahbazi, A; Wang, L; Ibrahim, S; Worku, M; Schimmel, K

2012-01-01

17

Genome Sequence of Lactococcus lactis subsp. lactis NCDO 2118, a GABA-Producing Strain.  

PubMed

Lactococcus lactis subsp. lactis NCDO 2118 is a nondairy lactic acid bacterium, a xylose fermenter, and a gamma-aminobutyric acid (GABA) producer isolated from frozen peas. Here, we report the complete genome sequence of L.lactis NCDO 2118, a strain with probiotic potential activity. PMID:25278529

Oliveira, Letcia C; Saraiva, Tesslia D L; Soares, Siomar C; Ramos, Rommel T J; S, Pablo H C G; Carneiro, Adriana R; Miranda, Fbio; Freire, Matheus; Renan, Wendel; Jnior, Alberto F O; Santos, Anderson R; Pinto, Anne C; Souza, Bianca M; Castro, Camila P; Diniz, Carlos A A; Rocha, Clarissa S; Mariano, Diego C B; de Aguiar, Edgar L; Folador, Edson L; Barbosa, Eudes G V; Aburjaile, Flavia F; Gonalves, Lucas A; Guimares, Lus C; Azevedo, Marcela; Agresti, Pamela C M; Silva, Renata F; Tiwari, Sandeep; Almeida, Sintia S; Hassan, Syed S; Pereira, Vanessa B; Abreu, Vinicius A C; Pereira, Ulisses P; Dorella, Fernanda A; Carvalho, Alex F; Pereira, Felipe L; Leal, Carlos A G; Figueiredo, Henrique C P; Silva, Artur; Miyoshi, Anderson; Azevedo, Vasco

2014-01-01

18

Genome Sequence of Lactococcus lactis subsp. lactis NCDO 2118, a GABA-Producing Strain  

PubMed Central

Lactococcus lactis subsp. lactis NCDO 2118 is a nondairy lactic acid bacterium, a xylose fermenter, and a gamma-aminobutyric acid (GABA) producer isolated from frozen peas. Here, we report the complete genome sequence of L.lactis NCDO 2118, a strain with probiotic potential activity. PMID:25278529

Oliveira, Letcia C.; Saraiva, Tesslia D. L.; Soares, Siomar C.; Ramos, Rommel T. J.; S, Pablo H. C. G.; Carneiro, Adriana R.; Miranda, Fbio; Freire, Matheus; Renan, Wendel; Jnior, Alberto F. O.; Santos, Anderson R.; Pinto, Anne C.; Souza, Bianca M.; Castro, Camila P.; Diniz, Carlos A. A.; Rocha, Clarissa S.; Mariano, Diego C. B.; de Aguiar, Edgar L.; Folador, Edson L.; Barbosa, Eudes G. V.; Aburjaile, Flavia F.; Gonalves, Lucas A.; Guimares, Lus C.; Azevedo, Marcela; Agresti, Pamela C. M.; Silva, Renata F.; Tiwari, Sandeep; Almeida, Sintia S.; Hassan, Syed S.; Pereira, Vanessa B.; Abreu, Vinicius A. C.; Pereira, Ulisses P.; Dorella, Fernanda A.; Carvalho, Alex F.; Pereira, Felipe L.; Leal, Carlos A. G.; Figueiredo, Henrique C. P.; Silva, Artur; Miyoshi, Anderson

2014-01-01

19

Dissolution of Xylose Metabolism in Lactococcus lactis  

Microsoft Academic Search

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus

KARN A. ERLANDSON; JOO-HEON PARK; WISSAM EL KHAL; El Khal; H.-H. Kao; P. Basaran; S. Brydges; C. A. Batt

2000-01-01

20

BactPepDB: a database of predicted peptides from a exhaustive survey of complete prokaryote genomes  

PubMed Central

With the recent progress in complete genome sequencing, mining the increasing amount of genomic information available should in theory provide the means to discover new classes of peptides. However, annotation pipelines often do not consider small reading frames likely to be expressed. BactPepDB, available online at http://bactpepdb.rpbs.univ-paris-diderot.fr, is a database that aims at providing an exhaustive re-annotation of all complete prokaryotic genomeschromosomal and plasmid DNAavailable in RefSeq for coding sequences ranging between 10 and 80 amino acids. The identified peptides are classified as (i) previously identified in RefSeq, (ii) entity-overlapping (intragenic) or intergenic, and (iii) potential pseudogenesintergenic sequences corresponding to a portion of a previously annotated larger gene. Additional information is related to homologs within order, predicted signal sequence, transmembrane segments, disulfide bonds, secondary structure, and the existence of a related 3D structure in the Protein Databank. As a result, BactPepDB provides insights about candidate peptides, and provides information about their conservation, together with some of their expected biological/structural features. The BactPepDB interface allows to search for candidate peptides in the database, or to search for peptides similar to a query, according to the multiple properties predicted or related to genomic localization. Database URL: http://www.yeastgenome.org/ PMID:25377257

Rey, Julien; Deschavanne, Patrick; Tuffery, Pierre

2014-01-01

21

Extended-Spectrum ?-lactamase (ESBL) producing Enterobacter aerogenes phenotypically misidentified as Klebsiella pneumoniae or K. terrigena  

PubMed Central

Background Enterobacter aerogenes and Klebsiella pneumoniae are common isolates in clinical microbiology and important as producers of extended spectrum ?-lactamases (ESBL). The discrimination between both species, which is routinely based on biochemical characteristics, is generally accepted to be straightforward. Here we report that genotypically unrelated strains of E. aerogenes can be misidentified as K. pneumoniae by routine laboratories using standard biochemical identification and using identification automates. Results Ten clinical isolates, identified as K. pneumoniae or K. terrigena with the routinely used biochemical tests and with API-20E, were identified as E. aerogenes by tDNA-PCR an identification that was confirmed by 16S rRNA gene sequencing for five of these isolates. Misidentification also occurred when using the automated identification systems Vitek 2 and Phoenix, and was due to delayed positivity for ornithine decarboxylase and motility. Subculture and prolonged incubation resulted in positive results for ornithine decarboxylase and for motility. It could be shown by RAPD-analysis that the E. aerogenes strains belonged to different genotypes. Conclusions Clinical E. aerogenes isolates can be easily misidentified as Klebsiella due to delayed positivity for ornithine decarboxylase and motility. The phenomenon may be widespread, since it was shown to occur among genotypically unrelated strains from different hospitals and different isolation dates. A useful clue for correct identification is the presence of an inducible ?-lactamase, which is highly unusual for K. pneumoniae. In several instances, the use of genotypic techniques like tDNA-PCR may circumvent problems of phenotypic identification. PMID:15619329

Claeys, Geert; De Baere, Thierry; Wauters, Georges; Vandecandelaere, Patricia; Verschraegen, Gerda; Muylaert, An; Vaneechoutte, Mario

2004-01-01

22

The nac (nitrogen assimilation control) gene from Klebsiella aerogenes.  

PubMed Central

The Klebsiella aerogenes nac gene, whose product is necessary for nitrogen regulation of a number of operons, was identified and its DNA sequence determined. The nac sequence predicted a protein a 305 amino acids with a strong similarity to members of the LysR family of regulatory proteins, especially OxyR from Escherichia coli. Analysis of proteins expressed in minicells showed that nac is a single-gene operon whose product has an apparent molecular weight of about 32 kDa as measured in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immediately downstream from nac is a two-gene operon, the first gene of which encodes another member of the LysR family. Upstream from nac is a tRNAAsn gene transcribed divergently from nac. About 60 bp upstream from the nac open reading frame lies a sequence nearly identical to the consensus for sigma 54-dependent promoters, with the conserved GG and GC nucleotides at -26 and -14 relative to the start of transcription. About 130 bp farther upstream (at -153 relative to the start of transcription) is a sequence nearly identical to the transcriptional activator NTRC-responsive enhancer consensus. Another weaker NTRC-binding site is located adjacent to this site (at -133 relative to the start of transcription). Thus, we propose that nac is transcribed by RNA polymerase carrying sigma 54 in response to the nitrogen regulatory (NTR) system. A transposon located between the promoter and the nac ORF prevented NTR-mediated expression of nac, supporting this identification of the promoter sequence. The insertion of over 5 kb of transposon DNA between the enhancer and its target promoter had only a weak effect on enhancer-mediated regulation, suggesting that enhancers may be able to act at a considerable distance on the bacterial chromosome. Images PMID:8458853

Schwacha, A; Bender, R A

1993-01-01

23

Comparative Phenotypic and Molecular Genetic Profiling of Wild Lactococcus lactis subsp. lactis Strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris Genotypes, Isolated from Starter-Free Cheeses Made of Raw Milk?  

PubMed Central

Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the cremoris genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely, atpA, rpoA, pheS, pepN, bcaT, pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the cremoris or lactis genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the cremoris group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies in this study appear to be good starter candidates. PMID:21666023

Fernndez, Elena; Alegra, ngel; Delgado, Susana; Martn, M. Cruz; Mayo, Baltasar

2011-01-01

24

Induction by Klebsiella aerogenes of a Melanin-Like Pigment in Cryptococcus neoformans  

PubMed Central

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment. PMID:16461709

Frases, Susana; Chaskes, Stuart; Dadachova, Ekaterina; Casadevall, Arturo

2006-01-01

25

Differentiation of Escherichia coil and Aerobacter aerogenes by Gas Liquid Chromatography  

Microsoft Academic Search

Six isolates of Aerobacter aerogenes; one Pseudomonas aergenoidcs ; five Escherichia coli; one Escherichia freundii; and one Escherichia intermedia were obtained from different sources and classified according to Bergey's 5(anual. Cultures of each were prepared in heat- and vacuum-treated milk and incubated 30 hr at 35 C. An attempt was made to differentiate these species by gas chromatography (GLC) of

R. E. BAWDON; R. BASSETTE

26

An adaptive response of Enterobacter aerogenes to imipenem: regulation of porin balance in clinical isolates.  

PubMed

Imipenem (IPM) is a carbapenem antibiotic frequently used in severe hospital infections. Several reports have mentioned the emergence of resistant isolates exhibiting membrane modifications. A study was conducted between September 2005 and August 2007 to survey infections due to Enterobacter aerogenes in patients hospitalised in a French university hospital. Resistant E. aerogenes clinical isolates obtained from patients treated with IPM and collected during the 3 months following initiation of treatment were phenotypically and molecularly characterised for ?-lactamases, efflux pumps activity and outer membrane proteins. Among the 339 patients infected with E. aerogenes during the study period, 41 isolates (12.1%) were resistant to extended-spectrum cephalosporins and 17 patients (5.0%) were treated with IPM. The isolates from these 17 patients presented TEM-24 and basal efflux expression. Following IPM treatment, an IPM-intermediate-susceptible (IPM-I) isolate emerged in 11 patients and an IPM-resistant (IPM-R) isolate in 6 patients. A change in the porin balance (Omp35/Omp36) was observed in IPM-I isolates exhibiting ertapenem resistance. Finally, a porin deficiency (Omp35 and Omp36 absence) was detected in IPM-R isolates associated with efflux pump expression. This study indicates that the alteration in porin expression, including the shift of porin expression and lack of porins, contribute to the E. aerogenes adaptive response to IPM treatment. PMID:23280442

Lavigne, Jean-Philippe; Sotto, Albert; Nicolas-Chanoine, Marie-Hlne; Bouziges, Nicole; Pags, Jean-Marie; Davin-Regli, Anne

2013-02-01

27

Lactococcus lactis subspecies lactis also causes white muscle disease in farmed giant freshwater prawns Macrobrachium rosenbergii.  

PubMed

From May to August 2001 in Taiwan, 27 farms for the giant freshwater prawn Macrobrachium rosenbergii experienced white tail disease outbreaks in animals approximately 3 to 5 mo old, with total lengths from 6 to 8 cm. Examination of the infected prawns revealed not only previously reported Lactococcus garvieae (16 farms) but also the novel L. lactis subsp. lactis (10 farms). One farm had shrimp infected with both bacteria. In the farms with L. lactis infections, the cumulative mortality was approximately 25 to 60%. Gross signs of disease were opaque and whitish muscles, while histopathology included marked edema and necrotic lesions, with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain/heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Eleven isolates from different farms were identified as L. lactis subsp. lactis using API 20 Strep and Rapid ID32 Strep tests and using PCR assays specific for the L. lactis subsp. lactis 16S rDNA gene (650 bp amplicon) and for the 16S to 23S rDNA interspacer region (380 bp amplicon). In addition, sequencing of the full 16S rDNA genes of 2 of the isolates (MR17 and MR26; GenBank Accession Numbers AF493058 and AF493057, respectively) revealed 99.9% identity between the isolates and 98.7% identity to several complete 16S rRNA sequences of L. lactis subsp. lactis at GenBank. Experimental infections with our isolates gave gross signs and histopathological changes similar to those seen in naturally infected prawns. The mean lethal dose of 4 isolates and the reference strain L. lactis subsp. lactis BCRC 10791 ranged from 4.2 x 10(6) to 2.5 x 10(7) colony-forming units prawn(-1), indicating virulence similar to that previously reported for L. garvieae. This is the first report confirming L. lactis subsp. lactis as a pathogen in juvenile and adult prawns from aquaculture. PMID:18429437

Wang, Pei-Chi; Lin, Yu-De; Liaw, Li-Ling; Chern, Red-Shiung; Chen, Shih-Chu

2008-03-01

28

Glycolysis and the regulation of glucose transport in Lactococcus lactis spp. lactis in batch and fed-batch culture  

Microsoft Academic Search

BACKGROUND: Despite the fact that many reports deal with glycolysis in Lactococcus lactis, there is not much information on the regulation of uptake of glucose itself. The aim of the present work was to investigate the effect of the glucose level on its specific uptake rate. RESULTS: Studies on aeration levels in pH controlled L. lactis spp. lactis batch cultures

Maria Papagianni; Nicholaos Avramidis; George Filiousis

2007-01-01

29

Characteristics of hydrogen production of an Enterobacter aerogenes mutant generated by a new atmospheric and room temperature plasma (ARTP)  

Microsoft Academic Search

A novel atmospheric and room temperature plasma (ARTP) which used helium as the working gas was employed to generate mutants of Enterobacter aerogenes for improving the hydrogen production. For the mutation, 50?l of the E. aerogenes culture (OD600=2.0) was dipped onto a sterilized stainless steel plate (5mm in diameter). The plate was then treated for 3min by ARTP at the

Yuan Lu; Liyan Wang; Kun Ma; Guo Li; Chong Zhang; Hongxin Zhao; Qiheng Lai; He-Ping Li; Xin-Hui Xing

30

Molecular Epidemiology of an Outbreak of Multidrug-Resistant Enterobacter aerogenesInfections and In Vivo Emergence of Imipenem Resistance  

Microsoft Academic Search

Moleculartypingwasusedtoinvestigateanoutbreakofinfectioncausedbymultidrug-resistantEnterobacter aerogenes (MREA) susceptible only to gentamicin and imipenem in an intensive care unit (ICU). Over a 9-month period, ciprofloxacin-resistant E. aerogenes isolates were isolated from 34 patients, or 4.1% of ICU admissions, compared with a baseline rate of 0.1% in the previous period (P< 0.001). Infection developed in 15 (44%) patients. In vivo emergence of imipenem resistance (MIC, 32

YVES DE GHELDRE; NICOLE MAES; FRANCIS ROST; RAFAEL DE RYCK; PHILIPPE CLEVENBERGH; JEAN LOUIS VINCENT; ANDMARC J. STRUELENS

1997-01-01

31

Tryptophan metabolism in Klebsiella aerogenes: regulation of the utilization of aromatic amino acids as sources of nitrogen.  

PubMed Central

Klebsiella aerogenes utilized aromatic amino acids as sole sources of nitrogen but not as sole sources of carbon. K. aerogenes abstracted the alpha-amino group of these compounds by transamination and excreted the arylpyruvate portions into the medium. When tryptophan was utilized as the sole source of nitrogen by K. aerogenes, indolepyruvate was excreted into the medium, where it polymerized non-enzymatically to form a brick red pigment. At least four separate aromatic aminotransferase activities were found in K. aerogenes. One activity (aromatic aminotransferase I) appeared to be solely responsible for the aminotransferase reaction necessary for the growth of K. aerogenes when tryptophan was the source of nitrogen; the loss of this activity by mutation (tut) prevented the growth of cells on media containing this and other aromatic amino acids. None of the other aminotransferase activities in the cells could substitute for aromatic aminotransferase in this regard. Tryptophan-dependent pigment formation in K. aerogenes was positively controlled by the intracellular level of glutamine synthetase. Nevertheless, the aromatic aminotransferase activity in cells varied less than 2-fold in response to 10-fold or greater changes in the levels of glutamine synthetase. Glutamine synthetase affected the ability of the cells to take up tryptophan from the medium. Images PMID:6109705

Paris, C G; Magasanik, B

1981-01-01

32

Purification and partial characterization of bacteriocin produced by Lactococcus lactis ssp. lactis LL171.  

PubMed

Lactic acid bacteria (LAB) are possessing ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this regard, novel bacteriocin compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was purified by ammonium sulphate precipitation, dialysis and gel filtration. The molecular weight (?3.4 kDa) of obtained bacteriocin was confirmed by SDS-PAGE, which revealed single peptide band. Molecular identification of LAB strain isolated from Tulum Cheese was conducted using 16S rDNA gene sequencing as Lactococcus lactis ssp. lactis LL171. The amino acid sequences (KKIDTRTGKTMEKTEKKIELSLKNMKTAT) of the bacteriocin from Lactococcus lactis ssp. lactis LL171 was found unique and novel than reported bacteriocins. Further, the bacteriocin was possessed the thermostable property and active at wide range of pH values from 1 to 11. Thus, bacteriocin reported in this study has the potential applications property as food preservative agent. PMID:22805947

Kumari, Archana; Akko, Nefise; Akelik, Mustafa

2012-04-01

33

Clinical and bacteriological study of nosocomial infections due to Enterobacter aerogenes resistant to imipenem.  

PubMed Central

Enterobacter aerogenes strains resistant to imipenem were isolated in 10 patients, 7 of whom had received imipenem-cilastatin. The strains were differentiated by biotype, antibiotype, and plasmid content. All of the strains overproduced a chromosomal cephalosporinase and lost a major outer membrane protein with a size of about 40 kDa. In 5 of the 10 patients, E. aerogenes strains resistant to extended-spectrum cephalosporin were isolated during the same stay. In three patients, the similarity between the imipenem-susceptible and -resistant strains suggests the occurrence of mutation and reversion in vivo. The combination imipenem-cilastatin has been critically important for use with multiresistant strains of Enterobacter spp., but its use increases the risk of selection of imipenem-resistant strains. Images PMID:8417016

de Champs, C; Henquell, C; Guelon, D; Sirot, D; Gazuy, N; Sirot, J

1993-01-01

34

Engineered Enterobacter aerogenes for efficient utilization of sugarcane molasses in 2,3-butanediol production.  

PubMed

Sugarcane molasses is considered to be a good carbon source for biorefinery due to its high sugar content and low price. Sucrose occupies more than half of the sugar in the molasses. Enterobacter aerogenes is a good host strain for 2,3-butanediol production, but its utilization of sucrose is not very efficient. To improve sucrose utilization in E. aerogenes, a sucrose regulator (ScrR) was disrupted from the genomic DNA. The deletion mutation increased the sucrose consumption rate significantly when sucrose or sugarcane molasses was used as a carbon source. The 2,3-butanediol production from sugarcane molasses by the mutant was enhanced by 60% in batch fermentation compared to that by the wild type strain. In fed-batch fermentation, 98.69 g/L of 2,3-butanediol production was achieved at 36 h. PMID:23644066

Jung, Moo-Young; Park, Bu-Soo; Lee, Jinwon; Oh, Min-Kyu

2013-07-01

35

Development of a low-cost medium for production of nisin from Lactococcus lactis subsp. lactis  

Microsoft Academic Search

The goal of this project was to develop a lower-cost medium for nisin production, so this bacteriocin could be used in a broader\\u000a range of industrial fermentation processes. The objectives included: (1) evaluating methods for controlling the inhibitory\\u000a effect of lactic acid produced during fermentation, and (2) comparing two inexpensive complex media for nisin production.\\u000a Lactococcus lactis subsp. lactis was

Charlene E. Wolf-Hall; William R. Gibbons; Nichole A. Bauer

2009-01-01

36

Effect of Iron Deprivation on the Production of Siderophores and Outer Membrane Proteins in Klebsiella aerogenes  

Microsoft Academic Search

The outer membrane (OM) protein profile of Klebsiella aerogenes grown in an iron rich chemically defined medium (Fe+CDM) showed three major proteins of 32.5, 35.5 and 39.0 kDal. The 35.5 and 39-0 kDal proteins were non-covalently associated with peptidoglycan. At least six new iron regulated outer membrane proteins (IRMP) of 69, 70, 73, 75, 78 and 83 kDal, which were

PAUL WILLIAMS; MICHAEL R. W. BROWN; PETER A. LAMBERT

1984-01-01

37

Inactivation of Enterobacter aerogenes in reconstituted skim milk by high- and low-frequency ultrasound.  

PubMed

The inactivation of Enterobacter aerogenes in skim milk using low-frequency (20kHz) and high-frequency (850kHz) ultrasonication was investigated. It was found that low-frequency acoustic cavitation resulted in lethal damage to E. aerogenes. The bacteria were more sensitive to ultrasound in water than in reconstituted skim milk having different protein concentrations. However, high-frequency ultrasound was not able to inactivate E. aerogenes in milk even when powers as high as 50W for 60min were used. This study also showed that high-frequency ultrasonication had no influence on the viscosity and particle size of skim milk, whereas low-frequency ultrasonication resulted in the decrease in viscosity and particle size of milk. The decrease in particle size is believed to be due to the breakup of the fat globules, and possibly to the cleavage of the ?-casein present at the surface of the casein micelles. Whey proteins were also found to be slightly affected by low-frequency ultrasound, with the amounts of ?-lactalbumin and ?-lactoglobulin slightly decreasing. PMID:24394387

Gao, Shengpu; Hemar, Yacine; Lewis, Gillian D; Ashokkumar, Muthupandian

2014-11-01

38

Novel antibacterial activity of lactococcus lactis subspecies lactis z11 isolated from zabady.  

PubMed

The purpose of this study was to select and characterize a probiotic bacterium with distinctive antimicrobial activities. In this respect, Lactococcus lactis subspecies lactis Z11 (L. lactis Z11) isolated from Zabady (Arabian yoghurt) inhibited other strains of lactic acid bacteria and some food-born pathogens including Listeria monocytogenes, Bacillus cereus and staphylococcus aureus. The inhibitory activity of cell free supernatant (CFS) of L. lactis Z11 isolated from zabady was lost by proteolytic enzymes, heat resistant. Consequently, the active substance(s) of CFS was characterized as a bacteriocin. This bacteriocin has been shown to consist of protein but has no lipidic or glucidic moieties in its active molecule. Its activity was stable in the pH range 2.0 to 7.0 and was not affected by organic solvents. The L. lactis Z11 bacteriocin was produced in CFS throughout the mide to the late exponential phase of growth of the producer organism and maximum bacteriocin production was obtained at initial pH 6.5 at incubation temperature of about 30C. PMID:24151453

Enan, Gamal; Abdel-Shafi, Seham; Ouda, Sahar; Negm, Sally

2013-09-01

39

Novel Antibacterial Activity of Lactococcus Lactis Subspecies Lactis Z11 Isolated from Zabady  

PubMed Central

The purpose of this study was to select and characterize a probiotic bacterium with distinctive antimicrobial activities. In this respect, Lactococcus lactis subspecies lactis Z11 (L. lactis Z11) isolated from Zabady (Arabian yoghurt) inhibited other strains of lactic acid bacteria and some food-born pathogens including Listeria monocytogenes, Bacillus cereus and staphylococcus aureus. The inhibitory activity of cell free supernatant (CFS) of L. lactis Z11 isolated from zabady was lost by proteolytic enzymes, heat resistant. Consequently, the active substance(s) of CFS was characterized as a bacteriocin. This bacteriocin has been shown to consist of protein but has no lipidic or glucidic moieties in its active molecule. Its activity was stable in the pH range 2.0 to 7.0 and was not affected by organic solvents. The L. lactis Z11 bacteriocin was produced in CFS throughout the mide to the late exponential phase of growth of the producer organism and maximum bacteriocin production was obtained at initial pH 6.5 at incubation temperature of about 30C. PMID:24151453

Enan, Gamal; Abdel-Shafi, Seham; Ouda, Sahar; Negm, Sally

2013-01-01

40

The Amino-Terminal 100 Residues of the Nitrogen Assimilation Control Protein (NAC) Encode All Known Properties of NAC from Klebsiella aerogenes and Escherichia coli  

Microsoft Academic Search

The nitrogen assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within the

WILSON B. MUSE; ROBERT A. BENDER

1999-01-01

41

Enhanced Human Lysozyme Production by Kluyveromyces lactis  

Microsoft Academic Search

An attempt to enhance recombinant human lysozyme production by Kluyveromyces lactis K7 was performed in this study. In this study, the production of recombinant human lysozyme was investigated using shake\\u000a flasks and bioreactor under different cultivation conditions. It was demonstrated that 25C could enhance human lysozyme\\u000a production when compared with other temperatures tested. This study also demonstrated that higher biomass

Eric Lu Huang; Ali Demirci

2009-01-01

42

Lepidic predominant adenocarcinoma with aerogenous spread of mucin in a young patient -- a case report.  

PubMed

We present a unique case of a 26 year-old female non-smoker who expired following treatment for presumed pneumonias. At autopsy, lepidic predominant adenocarcinoma with aerogenous spread of mucin without evidence of invasion, a rare diagnosis that previously would have fallen under the umbrella of "bronchioloalveolar carcinoma," was found. Histopathology showed mucin-secreting neoplastic cells lining the alveolar walls, as well as exfoliated and dense aggregates of mucinous debris filling the alveoli. The immediate cause of death was respiratory failure, most likely due to the significant amount of tumor-produced mucin that filled the alveolar spaces, which literally drowned the patient. PMID:24768586

Nguyen, Elise; Hakimi, Matthew; Chavoshan, Bahman; Stringer, William; French, Samuel W

2014-06-01

43

Parameter Estimation of Actuators for Benchmark Active Control Technology (BACT) Wind Tunnel Model with Analysis of Wear and Aerodynamic Loading Effects  

NASA Technical Reports Server (NTRS)

This report describes the development of transfer function models for the trailing-edge and upper and lower spoiler actuators of the Benchmark Active Control Technology (BACT) wind tunnel model for application to control system analysis and design. A simple nonlinear least-squares parameter estimation approach is applied to determine transfer function parameters from frequency response data. Unconstrained quasi-Newton minimization of weighted frequency response error was employed to estimate the transfer function parameters. An analysis of the behavior of the actuators over time to assess the effects of wear and aerodynamic load by using the transfer function models is also presented. The frequency responses indicate consistent actuator behavior throughout the wind tunnel test and only slight degradation in effectiveness due to aerodynamic hinge loading. The resulting actuator models have been used in design, analysis, and simulation of controllers for the BACT to successfully suppress flutter over a wide range of conditions.

Waszak, Martin R.; Fung, Jimmy

1998-01-01

44

Immunological study of the regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes.  

PubMed

Regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes was analyzed by immunological techniques. Antibody directed against the purified arylsulfatase from K. aerogenes W70 was obtained from rabbits and characterized by immunoelectrophoresis, double-diffusion, quantitative precipitation, and enzyme neutralization tests. Arylsulfatase was located in the periplasmic space when the wild-type strain was cultured with methionine or with inorganic sulfate plus tyramine, but not with inorganic sulfate without tyramine, as the sole sulfur source. Tyramine oxidase was retained in the membrane fraction prepared from cells grown in the presence of tyramine. Arylsulfatase protein was not synthesized in the presence of tyramine and inorganic sulfate by mutant K611, which is deficient in tyramine oxidase (tynA). We conclude that the expression of the arylsulfatase gene (atsA) is regulated by the expression of tynA and that inorganic sulfate serves as a corepressor. In addition, strains mutated in the atsA gene were analyzed by using antibody. PMID:72063

Murooka, Y; Yamada, T; Tanabe, S; Harada, T

1977-10-01

45

Immunological study of the regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes.  

PubMed Central

Regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes was analyzed by immunological techniques. Antibody directed against the purified arylsulfatase from K. aerogenes W70 was obtained from rabbits and characterized by immunoelectrophoresis, double-diffusion, quantitative precipitation, and enzyme neutralization tests. Arylsulfatase was located in the periplasmic space when the wild-type strain was cultured with methionine or with inorganic sulfate plus tyramine, but not with inorganic sulfate without tyramine, as the sole sulfur source. Tyramine oxidase was retained in the membrane fraction prepared from cells grown in the presence of tyramine. Arylsulfatase protein was not synthesized in the presence of tyramine and inorganic sulfate by mutant K611, which is deficient in tyramine oxidase (tynA). We conclude that the expression of the arylsulfatase gene (atsA) is regulated by the expression of tynA and that inorganic sulfate serves as a corepressor. In addition, strains mutated in the atsA gene were analyzed by using antibody. Images PMID:72063

Murooka, Y; Yamada, T; Tanabe, S; Harada, T

1977-01-01

46

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis.  

PubMed Central

The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the alpha-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway. PMID:9335274

Goupil-Feuillerat, N; Cocaign-Bousquet, M; Godon, J J; Ehrlich, S D; Renault, P

1997-01-01

47

Influence of the carbon source on nisin production in Lactococcus lactis subsp. lactis batch fermentations  

Microsoft Academic Search

Nisin production by Lactucmcus lrrctis subsp. lactis NIZO 22186 was studied in batch fermentation using a complex medium. Nisin production showed primary metabolite kinetics: nisin biosynthesis took place during the active growth phase and completely stopped when cells entered the stationary phase. A stringent correlation could be observed between the expression of the prenisin gene (nisA) and the synthesis of

LUC DE VUYST; ERICK J. VANDAMME

1992-01-01

48

Detection and Viability of Lactococcus lactis throughout Cheese Ripening  

PubMed Central

Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese. PMID:25503474

Cocolin, Luca

2014-01-01

49

Metabolic and energetic aspects of the growth of Klebsiella aerogenes NCTC 418 on glucose in anaerobic chemostat culture  

Microsoft Academic Search

Klebsiella aerogenes NCTC 418 was cultured anaerobically in chemostat cultures (pH 6.8; 35 C) under carbon, phosphate-, ammonia-, sulphate- and potassium-limited conditions with glucose as the sole carbon- and energy source. The rates of uptake of glucose and excretion of fermentation products were quantitatively determined and carbon, hydrogen- and oxygen balances were constructed with recoveries better than 90%.

M. J. Teixeira de Mattos; D. W. Tempest

1983-01-01

50

The Influence of Dissolved Oxygen Concentration on the Respiration and Glucose Metabolism of Klebsiella aerogenes during Growth  

Microsoft Academic Search

SUMMARY The influence of dissolved oxygen concentration on the metabolism and respiration of growing Klebsiella aerogenes NCTC 8017 was studied by means of a continuous-flow culture technique. Different dissolved oxygen tensions (equivalent partial pressures) were obtained by varying the partial pressure of oxygen in the gas phase. The respiration rate (oxygen uptake rate per unit mass organism) was independent of

D. E. F. HARRISON; S. J. PIRT

1967-01-01

51

Modification of Outer Membrane Protein Profile and Evidence Suggesting an Active Drug Pump in Enterobacter aerogenes Clinical Strains  

Microsoft Academic Search

Two clinical strains of Enterobacter aerogenes that exhibited phenotypes of multiresistance to -lactam antibiotics, fluoroquinolones, chloramphenicol, tetracycline, and kanamycin were investigated. Both strains showed a porin pattern different from that of a susceptible strain, with a drastic reduction in the amount of the major porin but with an apparently conserved normal structure (size and immunogenicity), together with overproduction of two

Stephane Gayet; Renaud Chollet; Gerard Molle; Jean-Marie Pages

2003-01-01

52

Ornithine transport and exchange in Streptococcus lactis  

SciTech Connect

Resting cells of Streptococcus lactis 133 appeared to accumulate (/sup 14/C)ornithine to a high concentration in the absence of an exogenous energy source. However, analysis of intracellular amino acid pool constituents and results of transport experiments revealed that the accumulation of ornithine represented a homoexchange between extracellular (/sup 14/C)ornithine and unlabeled ornithine in the cell. The energy-independent exchange of ornithine was not inhibited by proton-conducting uncouplers or by metabolic inhibitors. Intracellular (/sup 14/C)ornithine was retained by resting cells after suspension in a buffered medium. However, addition of unlabeled ornithine to the suspension elicited rapid exit of labeled amino acid. The initial rate of exist of (/sup 14/C)ornithine was dependent on the concentration of unlabeled ornithine in the medium, but this accelerative exchange diffusion process caused no net loss of amino acid. By contrast, the presence of a fermentable energy source caused a rapid expulsion of and new decrease in the concentration of intracellular ornithine. Kinetic analyses of amino acid transport demonstrated competitive inhibition between lysine and ornithine, and data obtained by two-dimensional thin-layer chromatography established the heteroexchange of these basic amino acids. The effects of amino acids and of ornithine analogs on both entry and exit of (/sup 14/C)ornithine have been examined. The data suggest that common carrier mediates the entry and exchange of lysine, arginine, and ornithine in cells of S. lactis.

Thompson, J.

1987-09-01

53

Rewiring Lactococcus lactis for Ethanol Production  

PubMed Central

Lactic acid bacteria (LAB) are known for their high tolerance toward organic acids and alcohols (R. S. Gold, M. M. Meagher, R. Hutkins, and T. Conway, J. Ind. Microbiol. 10:4554, 1992) and could potentially serve as platform organisms for production of these compounds. In this study, we attempted to redirect the metabolism of LAB model organism Lactococcus lactis toward ethanol production. Codon-optimized Zymomonas mobilis pyruvate decarboxylase (PDC) was introduced and expressed from synthetic promoters in different strain backgrounds. In the wild-type L. lactis strain MG1363 growing on glucose, only small amounts of ethanol were obtained after introducing PDC, probably due to a low native alcohol dehydrogenase activity. When the same strains were grown on maltose, ethanol was the major product and lesser amounts of lactate, formate, and acetate were formed. Inactivating the lactate dehydrogenase genes ldhX, ldhB, and ldh and introducing codon-optimized Z. mobilis alcohol dehydrogenase (ADHB) in addition to PDC resulted in high-yield ethanol formation when strains were grown on glucose, with only minor amounts of by-products formed. Finally, a strain with ethanol as the sole observed fermentation product was obtained by further inactivating the phosphotransacetylase (PTA) and the native alcohol dehydrogenase (ADHE). PMID:23377945

Dehli, Tore; Jensen, Peter Ruhdal

2013-01-01

54

Bifidobacterium animalis subsp. lactis strains isolated from dog faeces.  

PubMed

The aim of the study was to identify and characterize dog bifidobacterial isolates and compare them with commercial probiotic strains. Sixteen isolates of Bifidobacterium animalis ssp. lactis from dog faeces (German Shepherd Dog) were identified by subspecies-specific PCR, MALDI-TOF MS and sequencing. This study is the first describing B. animalis ssp. lactis occurring within the intestinal tract of dogs. Our dog isolates showed slightly different fingerprinting profiles obtained by RAPD-PCR and REP-PCR from those isolated from yogurt and type strains of B. animalis ssp. lactis. Both, dog and yogurt origin strains indicated survival in the simulated in vitro digestion assay and were resistant to low pH and bile salts. Moreover, strong auto-aggregation activity was observed only in dog origin B. animalis ssp. lactis strains. Dog strains showed good properties predicting their survival ability in GIT and could be tested as a potential new probiotics for dogs or other hosts. PMID:22749610

Buneov, V?ra; Vlkov, Eva; Rada, Vojt?ch; Ro?kov, Srka; Svobodov, Ivona; Jebav, Luk; Kme?, Vladimr

2012-12-01

55

Microencapsulation of Bifidobacterium lactis for incorporation into soft foods  

Microsoft Academic Search

Summary Micro-encapsulation of the probiotic micro-organism Bifidobacterium lactis isolated from a bio-yoghurt starter culture, was carried out using a mixture of hydrated gellan and xanthan gums. Rheological studies showed that the gum mix was suitable for encapsulation of B. lactis, for incorporation into soft foods\\/beverages. The gel behaved as a non-Newtonian material, and the flow curve fitted well to the

L. D. McMaster; S. A. Kokott

2005-01-01

56

Peptidoglycan N-acetylglucosamine deacetylation decreases autolysis in Lactococcus lactis.  

PubMed

The gene xynD (renamed pgdA) of Lactococcus lactis IL1403 was shown to encode a peptidoglycan N-acetylglucosamine deacetylase. Inactivation of pgdA in L. lactis led to fully acetylated peptidoglycan, whereas cloning of pgdA on a multicopy plasmid vector resulted in an increased degree of peptidoglycan deacetylation, as shown by analysis of peptidoglycan constituent muropeptides. An increased amount of N-unsubstituted glucosamine residues in peptidoglycan resulted in a reduction of the rate of autolysis of L. lactis cells. The activity of the L. lactis major autolysin AcmA was tested on L. lactis cells or peptidoglycan with different degrees of de-N-acetylation. Deacetylated peptidoglycan exhibited decreased susceptibility to AcmA hydrolysis. This reduced susceptibility to AcmA did not result from reduced AcmA binding to peptidoglycan with an increasing degree of de-N-acetylation. In conclusion, enzymic N-acetylglucosamine deacetylation protects peptidoglycan from hydrolysis by the major autolysin AcmA in L. lactis cells, and this leads to decreased cellular autolysis. PMID:17906127

Meyrand, Mickael; Boughammoura, Ada; Courtin, Pascal; Mzange, Christine; Guillot, Alain; Chapot-Chartier, Marie-Pierre

2007-10-01

57

Overflow metabolism during anaerobic growth of Klebsiella aerogenes NCTC 418 on glycerol and dihydroxyacetone in chemostat culture  

Microsoft Academic Search

Klebsiella aerogenes NCTC 418 was grown anaerobically in chemostat culture with glycerol as source of carbon and energy. Glycerol-limited cultures did not ferment the carbon source with maximal efficiency but produced considerable amounts of 1,3-propanediol. The fraction of glycerol converted to this product depended on the growth rate and on the limitation: faster growing cells produced relatively more of this

H. Streekstra; M. J. Teixeira de Mattos; O. M. Neijssel; D. W. Tempest

1987-01-01

58

Repression of Glutamate Dehydrogenase Formation in Klebsiella aerogenes Requires Two Binding Sites for the Nitrogen Assimilation Control Protein, NAC  

Microsoft Academic Search

In Klebsiella aerogenes, the gdhA gene codes for glutamate dehydrogenase, one of the enzymes responsible for assimilating ammonia into glutamate. Expression of a gdhAp-lacZ transcriptional fusion was strongly re- pressed by the nitrogen assimilation control protein, NAC. This strong repression (>50-fold under conditions of severe nitrogen limitation) required the presence of two separate NAC binding sites centered at 89 and

Thomas J. Goss; Brian K. Janes; Robert A. Bender

2002-01-01

59

Co-fermentation of carbon sources by Enterobacter aerogenes ATCC 29007 to enhance the production of bioethanol.  

PubMed

We investigated the enhancement of bioethanol production in Enterobacter aerogenes ATCC 29007 by co-fermentation of carbon sources such as glycerol, glucose, galactose, sucrose, fructose, xylose, starch, mannitol and citric acid. Biofuel production increases with increasing growth rate of microorganisms; that is why we investigated the optimal growth rate of E. aerogenes ATCC 29007, using mixtures of different carbon sources with glycerol. E. aerogenes ATCC 29007 was incubated in media containing each carbon source and glycerol; growth rate and bioethanol production improved in all cases compared to those in medium containing glycerol alone. The growth rate and bioethanol production were highest with mannitol. Fermentation was carried out at 37 C for 18 h, pH 7, using 50 mL defined production medium in 100 mL serum bottles at 200 rpm. Bioethanol production under optimized conditions in medium containing 16 g/L mannitol and 20 g/L glycerol increased sixfold (32.10 g/L) than that containing glycerol alone (5.23 g/L) as the carbon source in anaerobic conditions. Similarly, bioethanol production using free cells in continuous co-fermentation also improved (27.28 g/L) when 90.37 % of 16 g/L mannitol and 67.15 % of 20 g/L glycerol were used. Although naturally existing or engineered microorganisms can ferment mixed sugars sequentially, the preferential utilization of glucose to non-glucose sugars often results in lower overall yield and productivity of ethanol. Here, we present new findings in E. aerogenes ATCC 29007 that can be used to improve bioethanol production by simultaneous co-fermentation of glycerol and mannitol. PMID:24185706

Thapa, Laxmi Prasad; Lee, Sang Jun; Yang, Xiao Guang; Yoo, Hah Young; Kim, Sung Bong; Park, Chulhwan; Kim, Seung Wook

2014-06-01

60

moaR, a gene that encodes a positive regulator of the monoamine regulon in Klebsiella aerogenes.  

PubMed Central

We cloned and sequenced a Klebsiella aerogenes gene (moaR) for activation of arylsulfatase synthesis by tyramine. This gene was cloned by complementation of a K. aerogenes mutant in which tyramine fails to relieve the arylsulfatase repression caused by sulfur compounds. The moaR gene also activated induction of the synthesis of both tyramine oxidase and the 30-kDa protein that is specifically induced by high concentrations of tyramine or catecholamines. The moaR gene on the chromosome of the wild-type strain of K. aerogenes was disrupted by homologous recombination with a plasmid containing the inactivated moaR. The resultant mutant showed the same phenotype as previously isolated atsT mutant strains that are negative for the derepressed synthesis of arylsulfatase. In this mutant strain, tyramine also failed to induce the synthesis of tyramine oxidase or the production of a 30-kDa protein. The moaR gene is capable of encoding a protein of 26,238 Da. The putative MoaR protein has a helix-turn-helix motif in its C terminus. Thus, it seems likely that the MoaR protein regulates the operons by binding to the regulatory region of the monoamine regulon. The MoaR protein is subject to autogenous control, which was shown by use of a moaR'-lacZ transcriptional fusion. Images PMID:8407801

Azakami, H; Sugino, H; Yokoro, N; Iwata, N; Murooka, Y

1993-01-01

61

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.  

Code of Federal Regulations, 2014 CFR

...2014-04-01 2014-04-01 false Lactase enzyme preparation from Kluyveromyces lactis...Affirmed as GRAS 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the...

2014-04-01

62

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.  

Code of Federal Regulations, 2010 CFR

...2010-04-01 2009-04-01 true Lactase enzyme preparation from Kluyveromyces lactis...Affirmed as GRAS 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the...

2010-04-01

63

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.  

Code of Federal Regulations, 2013 CFR

...2013-04-01 2013-04-01 false Lactase enzyme preparation from Kluyveromyces lactis...Affirmed as GRAS 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the...

2013-04-01

64

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.  

Code of Federal Regulations, 2012 CFR

...2012-04-01 2012-04-01 false Lactase enzyme preparation from Kluyveromyces lactis...Affirmed as GRAS 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the...

2012-04-01

65

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.  

Code of Federal Regulations, 2011 CFR

...2011-04-01 2011-04-01 false Lactase enzyme preparation from Kluyveromyces lactis...Affirmed as GRAS 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the...

2011-04-01

66

Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production  

Microsoft Academic Search

Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow

Yves Le Loir; Vasco Azevedo; Sergio C Oliveira; Daniela A Freitas; Anderson Miyoshi; Luis G Bermdez-Humarn; Sbastien Nouaille; Luciana A Ribeiro; Sophie Leclercq; Jane E Gabriel; Maric N Oliveira; Cathy Charlier; Michel Gautier; Philippe Langella

2005-01-01

67

Genetic control of tyramine oxidase, which is involved in derepressed synthesis of arylsulfatase in Klebsiella aerogenes.  

PubMed Central

Mutants of Klebsiella aerogenes with three types of mutations affecting regulation of tyramine oxidase were isolated by a simple selection method. In the first type, the mutation (tynP) was closely linked to the structural gene for tyramine oxidase tynA). The order of mutation sites was atsA-tynP-tynA. In the second type, the mutation that relieves catabolite repression of the syntheses of several catabolite repression-sensitive enzymes are not linked to the tyn gene by P1 transduction. These strains contained high levels of cyclic adenosine 5'-monophosphate when grown on glucose. The third type of mutation, in which tyramine oxidase was synthesized constitutively, was shown by genetic analysis to involve mutations of tynP and tynR. The tynR gene was not linked to tynA. Results using the constitutive mutants showed that the constitutive expression of the tynA gene resulted in depression of arylsulfatase synthesis in the absence of tyramine. PMID:6249789

Oka, M; Murooka, Y; Harada, T

1980-01-01

68

Genetic control of tyramine oxidase, which is involved in derepressed synthesis of arylsulfatase in Klebsiella aerogenes.  

PubMed

Mutants of Klebsiella aerogenes with three types of mutations affecting regulation of tyramine oxidase were isolated by a simple selection method. In the first type, the mutation (tynP) was closely linked to the structural gene for tyramine oxidase tynA). The order of mutation sites was atsA-tynP-tynA. In the second type, the mutation that relieves catabolite repression of the syntheses of several catabolite repression-sensitive enzymes are not linked to the tyn gene by P1 transduction. These strains contained high levels of cyclic adenosine 5'-monophosphate when grown on glucose. The third type of mutation, in which tyramine oxidase was synthesized constitutively, was shown by genetic analysis to involve mutations of tynP and tynR. The tynR gene was not linked to tynA. Results using the constitutive mutants showed that the constitutive expression of the tynA gene resulted in depression of arylsulfatase synthesis in the absence of tyramine. PMID:6249789

Oka, M; Murooka, Y; Harada, T

1980-07-01

69

[Mechanisms of bioelectricity generation in Enterobacter aerogenes-based microbial fuel cells].  

PubMed

Microbial fuel cells (MFCs) using hydrogen-producing bacteria (HPB) could utilize a large number of substrates to generate power. However, the coulombic efficiency is limited by the fact that only suspended cells are used as biocatalyst in anodic medium. MFCs using Fe (III)-reducing bacteria have high energy recovery efficiency, but can only utilize some simple organic matters. In this study, Enterobacter aerogenes XM02, a hydrogen-producing strain with Fe(III)-reducing activity, was selected as biocatalyst for MFCs, which could produce electricity by digesting lots of carbohydrates even starch. Graphite felt, a material with high specific surface area and hydrogen catalysis, instead of carbon paper supported platinum, was used as anode material. The coulombic efficiency had been substantially improved from 1.68% to 42.49%, higher than other HPB-based MFCs previously reported. The SEM image proved the ability of XM02 strain to colonize on the anode surface. Power generation of MFCs could restore quickly when anodic medium was completely replaced with non-growth medium containing glucose. This suggested that the attached cells contributed to electricity production because planktonic cells had been removed during the medium replacement. This study proposed the mechanism of power generated from in situ oxidation of hydrogen produced by the XM02 strain biofilm. PMID:19545032

Zhang, Jin-Tao; Zhou, Shun-Gui; Zhang, Li-Xia; Lu, Na; Deng, Li-Fang; Ni, Jin-Ren

2009-04-15

70

The nitrogen assimilation control protein, NAC, is a DNA binding transcription activator in Klebsiella aerogenes.  

PubMed Central

A 32-kDa polypeptide corresponding to NAC, the product of the Klebsiella aerogenes nac gene, was overexpressed from a plasmid carrying a tac'-'nac operon fusion and purified to near homogeneity by taking advantage of its unusual solubility properties. NAC was able to shift the electrophoretic migration of DNA fragments carrying the NAC-sensitive promoters hutUp, putPp1, and ureDp. The interaction between NAC and hutUp was localized to a 26-bp region centered approximately 64 bp upstream of the hutUp transcription initiation site. Moreover, NAC protected this region from DNase I digestion. Mobility shift and DNase I protection studies utilizing the putP and ureD promoter regions identified NAC-binding regions of sizes and locations similar to those found in hutUp. Comparison of the DNA sequences which were protected from DNase I digestion by NAC suggests a minimal NAC-binding consensus sequence: 5'-ATA-N9-TAT-3'. In vitro transcription assays demonstrated that NAC was capable of activating the transcription of hutUp by sigma 70-RNA polymerase holoenzyme when this promoter was presented as either a linear or supercoiled DNA molecule. Thus, NAC displays the in vitro DNA-binding and transcription activation properties which have been predicted for the product of the nac gene. PMID:7768865

Goss, T J; Bender, R A

1995-01-01

71

Optimization of cultural conditions for conversion of glycerol to ethanol by Enterobacter aerogenes S012.  

PubMed

The aim of this research is to optimize the cultural conditions for the conversion of glycerol to ethanol by Enterobacter aerogenes S012. Taguchi method was used to screen the cultural conditions based on their signal to noise ratio (SN). Temperature (C), agitation speed (rpm) and time (h) were found to have the highest influence on both glycerol utilization and ethanol production by the organism while pH had the lowest. Full factorial design, statistical analysis, and regression model equation were used to optimize the selected cultural parameters for maximum ethanol production. The result showed that fermentation at 38C and 200 rpm for 48 h would be ideal for the bacteria to produce maximum amount of ethanol from glycerol. At these optimum conditions, ethanol production, yield and productivity were 25.4 g/l, 0.53 g/l/h, and 1.12 mol/mol-glycerol, repectively. Ethanol production increased to 26.5 g/l while yield and productivity decreased to 1.04 mol/mol-glycerol and 0.37 g/l/h, respectively, after 72 h. Analysis of the fermentation products was performed using HPLC, while anaerobic condition was created by purging the fermentation vessel with nitrogen gas. PMID:23388539

Nwachukwu, Raymond E S; Shahbazi, Abolghasem; Wang, Lijun; Worku, Mulumebet; Ibrahim, Salam; Schimmel, Keith

2013-01-01

72

In vivo inactivation of glycerol dehydrogenase in Klebsiella aerogenes: properties of active and inactivated proteins.  

PubMed Central

Glycerol:oxidized nicotinamide adenine dinucleotide (NAD+) 2-oxidoreductase (EC 1.1.1.6), an inducible enzyme for anaerobic glycerol catabolism in Klebsiella aerogenes, was purified and found to have a molecular weight of 79,000 by gel electrophoresis. The protein seemed to be enzymatically active either as a dimer of a 40,000-dalton peptide at pH 8.6 or as a tetramer of 160,000 molecular weight at pH 7.0. The enzyme activity was present at high levels in cells growing anaerobically on glycerol, but disappeared with a half-life of about 45 min if molecular oxygen was introduced to the culture. In contrast, no such phenomenon occurred with dihydroxyacetone kinase activity, the second enzyme in the pathway. Immunochemical analysis showed that the inactivation of the oxidoreductase did not involve degradation of the protein. Furthermore, subunits of the active and inactive forms of the enzyme were indistinguishable in size on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and had similar isoelectric points (pH 4.7). Inactivation did, however, alter the gel filtration properties of the enzyme protein and, more importantly, reduced its affinity for the dye Cibacron F3GA and the coenzyme NAD+. Images PMID:6988402

Ruch, F E; Lin, E C; Kowit, J D; Tang, C T; Goldberg, A L

1980-01-01

73

The oxygen level determines the fermentation pattern in Kluyveromyces lactis.  

PubMed

Yeasts belonging to the lineage that underwent whole-genome duplication (WGD) possess a good fermentative potential and can proliferate in the absence of oxygen. In this study, we analyzed the pre-WGD yeast Kluyveromyces lactis and its ability to grow under oxygen-limited conditions. Under these conditions, K. lactis starts to increase the glucose metabolism and accumulates ethanol and glycerol. However, under more limited conditions, the fermentative metabolism decreases, causing a slow growth rate. In contrast, Saccharomyces cerevisiae and Saccharomyces kluyveri in anaerobiosis exhibit almost the same growth rate as in aerobiosis. In this work, we showed that in K. lactis, under oxygen-limited conditions, a decreased expression of RAG1 occurred. The activity of glucose-6-phosphate dehydrogenase also decreased, likely causing a reduced flux in the pentose phosphate pathway. Comparison of related and characterized yeasts suggests that the behavior observed in K. lactis could reflect the lack of an efficient mechanism to maintain a high glycolytic flux and to balance the redox homeostasis under hypoxic conditions. This could be a consequence of a recent specialization of K. lactis toward living in a niche where the ethanol accumulation at high oxygen concentrations and the ability to survive at a low oxygen concentration do not represent an advantage. PMID:19500150

Merico, Annamaria; Galafassi, Silvia; Piskur, Jure; Compagno, Concetta

2009-08-01

74

Impact of osmotic stress on protein diffusion in Lactococcus lactis.  

PubMed

We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram-positive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582?kDa) and 12-transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ??1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram-positive and Gram-negative bacteria. PMID:25244659

Mika, Jacek T; Schavemaker, Paul E; Krasnikov, Victor; Poolman, Bert

2014-11-01

75

[Characteristics and identification of bacteriocins produced by Lactococcus lactis subsp. lactis 194-K].  

PubMed

The Lactococcus lactis subsp. lactis 194-K strain has been established to be able to produce two bacteriocins, one of which was identified as the known lantibiotic nisin A, and the other 194-D bacteriocin represents a polypeptide with a 2589-Da molecular mass and comprises 20 amino acid residues. Both bacteriocins were produced in varying proportions in all of the studied nutrient media, which support the growth of the producer. Depending on the cultivation medium, the nisin A content was 380- to 1123-fold lower in the 194-K stain culture fluid than that of the 194-D peptide. In comparision to to nisin A Bacteriocin 194-D possessed a wide range of antibacterial activity and suppressed the growth of both Gram-positive and Gram-negative bacteria. An optimal medium for 194-D bacteriocin synthesis was shown to be a fermentation medium which contained yeast extract, casein hydrolysate, and potassium phosphate. The biosynthesis ofbacteriocin 194-D by the 194-K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture fluid was observed at 14-20 h of the strain's growth. PMID:23330388

Ustiugova, E A; Timofeeva, A V; Stoianova, L G; Netrusov, A I; Katrukha, G S

2012-01-01

76

Stress response kinetics of two nisin producer strains of Lactococcus lactis spp. lactis.  

PubMed

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied. PMID:18769876

Sim?ek, Omer; Buzrul, Sencer; Akko, Nefise; Alpas, Hami; Akelik, Mustafa

2009-08-01

77

Probiotic characterization of potential hydrolases producing Lactococcus lactis subsp. lactis isolated from pickled yam.  

PubMed

The aim of this study was to characterize potential probiotic strain co-producing ?-amylase and ?-galactosidase. Sixty-three strains, isolated from pickle samples were screened for their hydrolase producing capacity by utilizing different starches as carbon source. One out of 63 strains, isolated from traditionally fermented pickled yam showing maximum hydrolase activity (?-amylase (36.9?U/ml) and ?-galactosidase (42.6?U/ml)) within a period of 48 hours was identified as Lactococcus lactis subsp. lactis. Further, it was assessed for the probiotic characteristics under gastrointestinal conditions like acidic, alkaline, proteolytic enzymes, bile stress and found to exhibit tolerance to these stresses. The therapeutic potential of the isolate is implicated because of its antagonistic effect against enteric foodborne pathogens (Salmonella typhimurium, Escherichia coli 0157:H7, Staphylococcus aureus, Yersinia enterocolitica and Aeromonas hydrophila). The results of this study entail a potential applicability of the isolate in developing future probiotic foods besides the production of industrially significant hydrolases. PMID:24020495

Bhanwar, Seema; Singh, Arashdeep; Ganguli, Abhijit

2014-02-01

78

Two Roles for the DNA Recognition Site of the Klebsiella aerogenes Nitrogen Assimilation Control Protein  

PubMed Central

The nitrogen assimilation control protein (NAC) binds to a site within the promoter region of the histidine utilization operon (hutUH) of Klebsiella aerogenes, and NAC bound at this site activates transcription of hutUH. This NAC-binding site was characterized by a combination of random and directed DNA mutagenesis. Mutations that abolished or diminished in vivo transcriptional activation by NAC were found to lie within a 15-bp region contained within the 26-bp region protected by NAC from DNase I digestion. This 15-bp core has the palindromic ends ATA and TAT, and it matches the consensus for LysR family transcriptional regulators. Protein-binding experiments showed that transcriptional activation in vivo decreased with decreasing binding in vitro. In contrast to the NAC-binding site from hutUH, the NAC-binding site from the gdhA promoter failed to activate transcription from a semisynthetic promoter, and this failure was not due to weak binding or greatly distorted protein-DNA structure. Mutations in the promoter-proximal half-site of the NAC-binding site from gdhA allowed this site to activate transcription. Similar studies using the NAC-binding site from hut showed that two mutations in the promoter proximal half-site increased binding but abolished transcriptional activation. Interestingly, for symmetric mutations in the promoter-distal half-site, loss of transcriptional activation was always correlated with a decrease in binding. We conclude from these observations that if the binding in vitro reflects the binding in vivo, then binding of NAC to DNA is not sufficient for transcriptional activation and that the NAC-binding site can be functionally divided in two half-sites, with related but different functions. PMID:9457860

Pomposiello, Pablo J.; Janes, Brian K.; Bender, Robert A.

1998-01-01

79

Molecular epidemiology of an outbreak of multidrug-resistant Enterobacter aerogenes infections and in vivo emergence of imipenem resistance.  

PubMed Central

Molecular typing was used to investigate an outbreak of infection caused by multidrug-resistant Enterobacter aerogenes (MREA) susceptible only to gentamicin and imipenem in an intensive care unit (ICU). Over a 9-month period, ciprofloxacin-resistant E. aerogenes isolates were isolated from 34 patients, or 4.1% of ICU admissions, compared with a baseline rate of 0.1% in the previous period (P < 0.001). Infection developed in 15 (44%) patients. In vivo emergence of imipenem resistance (MIC, 32 micrograms/ml) of organisms causing deep-seated infection was observed in two (13%) of these patients following prolonged therapy with imipenem and gentamicin. Arbitrarily primed PCR (AP-PCR) analysis with ERIC1R and ERIC2 primers and pulsed-field gel electrophoresis (PFGE) analysis of XbaI macrorestriction patterns concordantly showed that outbreak-associated MREA isolates were clonally related and distinct from epidemiologically unrelated strains. AP-PCR and PFGE showed discrimination indices of 0.88 and 0.98, respectively. Space-time clustering of cases within units suggests that the epidemic-related MREA isolates were transmitted on the hands of the health care personnel. A case-control study and repeated environmental culture surveys failed to identify a common source or procedure associated with transmission. In spite of the early implementation of isolation measures, the incidence of MREA colonization remained stable until all colonized patients were discharged. This study confirms the usefulness of AP-PCR and PFGE analyses for the epidemiological study of E. aerogenes and underscores the difficulty of controlling the spread of multiresistant clones of this organism in the ICU setting. The emergence of imipenem resistance represents a threat because virtually no therapeutic option is available for such strains. PMID:8968898

De Gheldre, Y; Maes, N; Rost, F; De Ryck, R; Clevenbergh, P; Vincent, J L; Struelens, M J

1997-01-01

80

Recombinant Lactococcus lactis fails to secrete bovine chymosine.  

PubMed

Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein. PMID:25184638

Luerce, Tesslia Diniz; Azevedo, Marcela Santiago Pacheco; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Pontes, Daniela

2014-09-01

81

Recombinant Lactococcus lactis fails to secrete bovine chymosine.  

PubMed

Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein. PMID:25482140

Luerce, Tesslia Diniz; Azevedo, Marcela Santiago Pacheco; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Pontes, Daniela

2014-09-01

82

Failure of the MicroScan WalkAway System To Detect Heteroresistance to Carbapenems in a Patient with Enterobacter aerogenes Bacteremia?  

PubMed Central

We report the failure of the automated MicroScan WalkAway system to detect carbapenem heteroresistance in Enterobacter aerogenes. Carbapenem resistance has become an increasing concern in recent years, and robust surveillance is required to prevent dissemination of resistant strains. Reliance on automated systems may delay the detection of emerging resistance. PMID:19641071

Gordon, N. C.; Wareham, D. W.

2009-01-01

83

Alanine Catabolism in Klebsiella aerogenes: Molecular Characterization of the dadAB Operon and Its Regulation by the Nitrogen Assimilation Control Protein  

Microsoft Academic Search

Klebsiella aerogenes strains with reduced levels of D-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory

BRIAN K. JANES; ROBERT A. BENDER

1998-01-01

84

Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes  

Microsoft Academic Search

Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were isolated, and their cleavage specificity and rate towards ?s1-and ?-casein was investigated. The catalytic properties of

Pieter Vos; Ingrid J. Boerrigter; Girbe Buist; Alfred J. Haandrikman; Monique Nijhuis; Marjon B. de Reuver; Roland J. Siezen; Gerard Venema; Willem M. de Vos; Jan Kok

1991-01-01

85

Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability  

SciTech Connect

Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.

Chopin, M.C.; Chopin, A.; Rouault, A.; Simon, D.

1986-02-01

86

Generation of Dipeptidyl Peptidase-IV-Inhibiting Peptides from ?-Lactoglobulin Secreted by Lactococcus lactis  

PubMed Central

Previous studies showed that hydrolysates of ?-lactoglobulin (BLG) prepared using gastrointestinal proteases strongly inhibit dipeptidyl peptidase-IV (DPP-IV) activity in vitro. In this study, we developed a BLG-secreting Lactococcus lactis strain as a delivery vehicle and in situ expression system. Interestingly, trypsin-digested recombinant BLG from L. lactis inhibited DPP-IV activity, suggesting that BLG-secreting L. lactis may be useful in the treatment of type 2 diabetes mellitus. PMID:25157356

Shigemori, Suguru; Oshiro, Kazushi; Wang, Pengfei; Yamamoto, Yoshinari; Wang, Yeqin; Sato, Takashi; Uyeno, Yutaka; Shimosato, Takeshi

2014-01-01

87

Genetic Evidence for a Defective Xylan Degradation Pathway in Lactococcus lactis  

Microsoft Academic Search

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immedi- ately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl.

KARN A. ERLANDSON; SOAZIG C. DELAMARRE; CARL A. BATT

2001-01-01

88

Expression of a chitinase gene from Serratia marcescens in Lactococcus lactis and Lactobacillus plantarum  

Microsoft Academic Search

A chitinase gene from the Gram-negative bacterium Serratia marcescens BJL200 was cloned in Lactococcus lactis subsp. lactis MG1363 and in the silage inoculum strain Lactobacillus plantarum E19b. The chitinase gene was expressed as an active enzyme at a low level in Lactococcus lactis, when cloned in the same transcriptional orientation as the gene specifying the replication protein of the vector

M. B. Brurberg; A. J. Haandrikman; K. J. Leenhouts; G. Venema; I. F. Nes

1994-01-01

89

Characterization of a Wild, Novel Nisin A-Producing Lactococcus Strain with an L. lactis subsp. cremoris Genotype and an L. lactis subsp. lactis Phenotype, Isolated from Greek Raw Milk  

PubMed Central

Several molecular taxonomic studies have revealed that many natural (wild) Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. lactis subspecies lactis cluster genotypically within subspecies cremoris and vice versa. Recently, we isolated two wild nisin-producing (Nis+) L. lactis strains, M78 and M104, of the lactis phenotype from Greek raw milk (J. Samelis, A. Lianou, A. Kakouri, C. Delbs, I. Rogelj, B. B. Matijaic, and M. C. Montel, J. Food Prot. 72:783790, 2009); strain M78 possess a novel nisin A sequence (GenBank accession number HM219853). In this study, the actual subspecies identity of M78 and M104 isolates was elucidated, using 16S rRNA and acmA (encoding lactococcal N-acetylmuramidase) gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh (encoding lactate dehydrogenase) gene phylogenies. Except the acmA gene analysis, molecular tools revealed that isolates M78 and M104 clustered with strains of the cremoris genotype, including the LMG 6897T strain, while they were distant from strains of the lactis genotype, including the LMG 6890T strain. The two wild isolates had identical repetitive sequence-based PCR (rep-PCR), randomly amplified polymorphic DNA (RAPD), plasmid, and whole-cell protein profiles and shared high 16S rRNA (99.9%) and ldh (100%) gene sequence homologies. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG 6890T strain. To our knowledge, this is the first complete identification report on a wild L. lactis subsp. cremoris genotype of the lactis phenotype which is capable of nisin A production and, thus, has strong potential for use as a novel dairy starter and/or protective culture. PMID:23542625

Parapouli, Maria; Delbs-Paus, Cline; Kakouri, Athanasia; Koukkou, Anna-Irini; Montel, Marie-Christine

2013-01-01

90

Cloning and nucleotide sequence of a negative regulator gene for Klebsiella aerogenes arylsulfatase synthesis and identification of the gene as folA.  

PubMed Central

A negative regulator gene for synthesis of arylsulfatase in Klebsiella aerogenes was cloned. Deletion analysis showed that the regulator gene was located within a 1.6-kb cloned segment. Transfer of the plasmid, which contains the cloned fragment, into constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsR; the synthesis of arylsulfatase was repressed in the presence of inorganic sulfate or cysteine, and this repression was relieved, in each case, by the addition of tyramine. The nucleotide sequence of the 1.6-kb fragment was determined. From the amino acid sequence deduced from the DNA sequence, we found two open reading frames. One of them lacked the N-terminal region but was highly homologous to the gene which codes for diadenosine tetraphosphatase (apaH) in Escherichia coli. The other open reading frame was located counterclockwise to the apaH-like gene. This gene was highly homologous to the gene which codes for dihydrofolate reductase (folA) in E. coli. We detected 30 times more activity of dihydrofolate reductase in the K. aerogenes strains carrying the plasmid, which contains the arylsulfatase regulator gene, than in the strains without plasmid. Further deletion analysis showed that the K. aerogenes folA gene is consistent with the essential region required for the repression of arylsulfatase synthesis. Transfer of a plasmid containing the E. coli folA gene into atsR mutant cells of K. aerogenes resulted in repression of the arylsulfatase synthesis. Thus, we conclude that the folA gene codes a negative regulator for the ats operon. PMID:1551851

Azakami, H; Sugino, H; Murooka, Y

1992-01-01

91

Insertional Mutagenesis To Generate Lantibiotic Resistance in Lactococcus lactis?  

PubMed Central

While the potential emergence of food spoilage and pathogenic bacteria with resistance to lantibiotics is a concern, the creation of derivatives of starter cultures and adjuncts that can grow in the presence of these antimicrobials may have applications in food fermentations. Here a bank of Lactococcus lactis IL1403 mutants was created and screened, and a number of novel genetic loci involved in lantibiotic resistance were identified. PMID:17526796

Guinane, Caitriona M.; Cotter, Paul D.; Lawton, Elaine M.; Hill, Colin; Ross, R. Paul

2007-01-01

92

Expanding the recombinant protein quality in Lactococcus lactis.  

PubMed

Background Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement.ResultsWe have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism.ConclusionsMetabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an excellent producer of recombinant proteins but also reveal room for significant improvement by the exploitation of external protein quality modulators. PMID:25471301

Cano-Garrido, Olivia; Rueda, Fabian L; Snchez-Garca, Laura; Ruiz-vila, Luis; Bosser, Ramon; Villaverde, Antonio; Garca-Fruits, Elena

2014-12-01

93

Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction  

Microsoft Academic Search

The post-translationally modified, antimicrobial peptide nisin is secreted by strains of Lactococcus lactis that contain the chromosomally located nisin biosynthetic gene cluster nisABTCIPRKFEG. When a 4-base pair deletion is introduced into the structural nisA gene (?nisA), transcription of ?nisA is abolished. Transcription of the ?nisA gene is restored by adding subinhibitory amounts of nisin, nisin mutants, or nisin analogs to

Oscar P. Kuipers; Marke M. Beerthuyzen; Evert J. Luesink; Willem M. de Vos

1995-01-01

94

Multivitamin production in Lactococcus lactis using metabolic engineering.  

PubMed

The dairy starter bacterium Lactococcus lactis has the potential to synthesize both folate (vitamin B11) and riboflavin (vitamin B2). By directed mutagenesis followed by selection and metabolic engineering we have modified two complicated biosynthetic pathways in L. lactis resulting in simultaneous overproduction of both folate and riboflavin: Following exposure to the riboflavin analogue roseoflavin we have isolated a spontaneous mutant of L. lactis strain NZ9000 that was changed from a riboflavin consumer into a riboflavin producer. This mutant contained a single base change in the regulatory region upstream of the riboflavin biosynthetic genes. By the constitutive overproduction of GTP cyclohydrolase I in this riboflavin-producing strain, the production of folate was increased as well. Novel foods, enriched through fermentation using these multivitamin-producing starters, could compensate the B-vitamin-deficiencies that are common even in highly developed countries and could specifically be used in dietary foods for the large fraction of the Caucasian people (10-15%) with mutations in the methylene tetrahydrofolate reductase (MTHFR). PMID:15113564

Sybesma, Wilbert; Burgess, Catherine; Starrenburg, Marjo; van Sinderen, Douwe; Hugenholtz, Jeroen

2004-04-01

95

LYS2 gene and its mutation in Kluyveromyces lactis.  

PubMed

The KlLYS2 gene, encoding the alpha-aminoadipate reductase of Kluyveromyces lactis, was isolated by complementation of a lysA1 mutant. The deduced amino acid sequence shared an identity of 73% with the LYS2 product of Saccharomyces cerevisiae. Despite the high sequence homology of the alpha-aminoadipate reductase genes, the two yeast species differently responded to the presence of alpha-aminoadipate in the medium. Wild-type S. cerevisiae is known to be sensitive to alpha-aminoadipate, but becomes resistant when mutated to lys2. In contrast, K. lactis strains were found to be naturally resistant to alpha-aminoadipate. Therefore, the positive selection procedure for the isolation of lys2 mutants on alpha-aminoadipate, as practised in S. cerevisiae, cannot be applied to K. lactis. A possible reason of this difference may be that the catalytic rate of the alpha-aminoadipate reductase differs in the two yeasts. The EMBL/Genbank Accession No. for the KlLYS2 gene is AJ504405. PMID:14587101

Alberti, Adriana; Ferrero, Iliana; Lodi, Tiziana

2003-10-30

96

RAG4 gene encodes a glucose sensor in Kluyveromyces lactis.  

PubMed Central

The rag4 mutant of Kluyveromyces lactis was previously isolated as a fermentation-deficient mutant, in which transcription of the major glucose transporter gene RAG1 was affected. The wild-type RAG4 was cloned by complementation of the rag4 mutation and found to encode a protein homologous to Snf3 and Rgt2 of Saccharomyces cerevisiae. These two proteins are thought to be sensors of low and high concentrations of glucose, respectively. Rag4, like Snf3 and Rgt2, is predicted to have the transmembrane structure of sugar transporter family proteins as well as a long C-terminal cytoplasmic tail possessing a characteristic 25-amino-acid sequence. Rag4 may therefore be expected to have a glucose-sensing function. However, the rag4 mutation was fully complemented by one copy of either SNF3 or RGT2. Since K. lactis appears to have no other genes of the SNF3/RGT2 type, we suggest that Rag4 of K. lactis may have a dual function of signaling high and low concentrations of glucose. In rag4 mutants, glucose repression of several inducible enzymes is abolished. PMID:11404320

Betina, S; Goffrini, P; Ferrero, I; Wsolowski-Louvel, M

2001-01-01

97

Structure of wild-type and mutant repressors and of the control region of the rbt operon of Klebsiella aerogenes.  

PubMed Central

Pentitol metabolism in Klebsiella aerogenes is encoded by continuous ribitol (rbt) and D-arabitol (dal) operons transcribed in bipolar fashion and sandwiched between long stretches of homologous DNA. The operons are separated by a central control region (2.2 kb) which encodes both the repressors and all the control sequences. The rbt repressor (270 amino acids) shows homology to the Escherichia coli lac repressor and other DNA-binding proteins. It is transcribed from the strand opposite the rbt operon and the intervening control region (254-bp) contains features which reflect the complex regulation. A rbt-constitutive mutant strain used in previous studies of experimental enzyme evolution encodes a truncated rbt-peptide of 133 residues due to a frameshift mutation. PMID:3891331

Wu, J; Anderton-Loviny, T; Smith, C A; Hartley, B S

1985-01-01

98

Epidemiological study of an outbreak due to multidrug-resistant Enterobacter aerogenes in a medical intensive care unit.  

PubMed Central

In 1993, 63 isolates of Enterobacter aerogenes were collected from 41 patients in a medical intensive care unit (ICU). During the same period, only 46 isolates from 32 patients were collected in the rest of the hospital. All isolates were analyzed by antibiotic resistance phenotype, and 77 representative isolates were differentiated by plasmid restriction analysis, ribotyping, and arbitrarily primed (AP)-PCR. The extended-spectrum beta-lactamases produced by 22 strains were characterized by determination of their isoelectric points and by hybridization of plasmid DNA with specific probes. The isolates were divided into 25 antibiotic resistance phenotypes, either susceptible (group I) or resistant (group II) to aminoglycosides, and exhibited three phenotypes of resistance to beta-lactams: chromosomally derepressed cephalosporinase alone or associated with either extended-spectrum beta-lactamases (mainly of the SHV-4 type) or imipenem resistance. The results of the tests divided the 77 representative isolates (group I, n = 21; group II, n = 56) into 15 plasmid profiles, 14 ribotypes, and 15 AP-PCR patterns. Although the resistant isolates (group II) exhibited different plasmid profiles, ribotyping and AP-PCR analysis demonstrated an identical chromosomal pattern, indicating an epidemiological relatedness. They were mainly found in the medical ICU and occasionally in other units. The susceptible strains (group I) had various and distinct markers and were mainly isolated in units other than the medical ICU. In conclusion, the presence of a nosocomial outbreak in an ICU and the spread of a multidrug-resistant epidemic strain throughout the hospital was confirmed. Ribotyping and AP-PCR represent discriminatory tools for the investigation of nosocomial outbreaks caused by E. aerogenes. PMID:8862578

Arpin, C; Coze, C; Rogues, A M; Gachie, J P; Bebear, C; Quentin, C

1996-01-01

99

Lactococcus lactis subsp. lactis infection in Bester sturgeon, a cultured hybrid of Huso huso Acipenser ruthenus, in Taiwan.  

PubMed

Approximately 5300 hybrid sturgeons with an average body weight of 600-800 g were farmed in 3 round tankers measuring 3m in diameter each containing 28,000 L of aerated groundwater. According to the owner's description, the diseased fish had anorexia, pale body color, and reddish spots on the abdomen. The morbidity and lethality rates in this outbreak were about 70% (3706/5300) and 100% (3706/3706), respectively. The clinical examination revealed enteritis, enlarged abdomen, and rapid respiration rate. The gross findings revealed a volume of about 4 mL of ascites. The histopathological examination showed multiple massive, hemorrhagic or coagulative necrotic foci in the liver and spleen. Furthermore, there was diffuse infiltration of glycogen in hepatic cells, and a few polymorphonuclear and mononuclear leucocytes were observed surrounding the spleen. Some bacterial clumps were noted around the necrotic foci. We also observed that there was moderate to severe, acute, multifocal, coagulative necrosis in the renal parenchyma, with some necrotic foci present beneath the margin of the kidney. Additionally, multifocal, coagulative necrosis was found in the pancreas. Results of microbiologic examinations, including biochemical characteristics, PCR amplification of 16S rRNA gene, sequencing and comparison, and phylogenetic analysis, revealed the pathogen of this infection was Lactococcus lactis subsp. lactis, and based on the results of an antimicrobial agent sensitivity test the bacterium was only sensitive to ampicillin and florfenicol. Additionally, results of in vivo experimental infections in hybrid tilapia showed that 110(8) and 110(9) CFU/mL of our isolate caused death in all fish and LD(50) values ranged from 10(2) to 10(5) CFU/mL. To the best of the authors' knowledge, this is the first reported case of Lactococcus lactis subsp. lactis infection in hybrid sturgeon. PMID:22098776

Chen, Ming-Hui; Hung, Shao-Wen; Shyu, Ching-Lin; Lin, Cheng-Chung; Liu, Pan-Chen; Chang, Chen-Hsuan; Shia, Wei-Yau; Cheng, Ching-Fu; Lin, Shiun-Long; Tu, Ching-Yu; Lin, Yu-Hsing; Wang, Way-Shyan

2012-10-01

100

Inactivation of the panE gene in Lactococcus lactis enhances formation of cheese aroma compounds.  

PubMed

Hydroxyacid dehydrogenases limit the conversion of ?-keto acids into aroma compounds. Here we report that inactivation of the panE gene, encoding the ?-hydroxyacid dehydrogenase activity in Lactococcus lactis, enhanced the formation of 3-methylbutanal and 3-methylbutanol. L. lactis IFPL953?panE was an efficient strain producing volatile compounds related to cheese aroma. PMID:23524675

de Cadianos, Luz P Gmez; Garca-Cayuela, Toms; Yvon, Mireille; Martinez-Cuesta, M Carmen; Pelez, Carmen; Requena, Teresa

2013-06-01

101

Inactivation of the panE Gene in Lactococcus lactis Enhances Formation of Cheese Aroma Compounds  

PubMed Central

Hydroxyacid dehydrogenases limit the conversion of ?-keto acids into aroma compounds. Here we report that inactivation of the panE gene, encoding the ?-hydroxyacid dehydrogenase activity in Lactococcus lactis, enhanced the formation of 3-methylbutanal and 3-methylbutanol. L. lactis IFPL953?panE was an efficient strain producing volatile compounds related to cheese aroma. PMID:23524675

de Cadianos, Luz P. Gmez; Garca-Cayuela, Toms; Yvon, Mireille; Martinez-Cuesta, M. Carmen; Pelez, Carmen

2013-01-01

102

A17, the First Sequenced Strain of Lactococcus lactis subsp. cremoris with Potential Immunomodulatory Functions  

PubMed Central

Lactococcus lactis subsp. cremoris A17, isolated from Taiwan fermented cabbage, is the first sequenced strain of L. lactis subsp. cremoris with immunomodulatory activity and antiallergic functions. The resulting A17 draft genome contains 2,679,936 bp and indicates that A17 is a potential exopolysaccharide-producing strain without any known virulence gene. PMID:25676767

Yang, Chih-Hsien; Wu, Chien-Chen; Cheng, Wei-Shen; Chung, Ming-Chuan

2015-01-01

103

Growth Enhancement of Bifidobacterium lactis Bo and Lactobacillus acidophilus Ki by Milk Hydrolyzates  

Microsoft Academic Search

The determination of the best conditions of prepa- ration of a (tentatively) probiotic starter culture that might be suitable for cheese making composed solely of Bifidobacterium lactis Bo and Lactobacillus acidophilus Ki is critical if a consistently reliable acid production is to be achieved, especially because bifidobacteria have stringent requirements for growth. Therefore, we determined whether B. lactis Bo and

Ana M. P. Gomes; F. Xavier Malcata; Frank A. M. Klaver

1998-01-01

104

A17, the First Sequenced Strain of Lactococcus lactis subsp. cremoris with Potential Immunomodulatory Functions.  

PubMed

Lactococcus lactis subsp. cremoris A17, isolated from Taiwan fermented cabbage, is the first sequenced strain of L. lactis subsp. cremoris with immunomodulatory activity and antiallergic functions. The resulting A17 draft genome contains 2,679,936 bp and indicates that A17 is a potential exopolysaccharide-producing strain without any known virulence gene. PMID:25676767

Yang, Chih-Hsien; Wu, Chien-Chen; Cheng, Wei-Shen; Chung, Ming-Chuan; Tsai, Ying-Chieh; Chang, Chuan-Hsiung

2015-01-01

105

Short communication: Genotypic and phenotypic identification of environmental streptococci and association of Lactococcus lactis ssp. lactis with intramammary infections among different dairy farms.  

PubMed

Lactococcus species are counted among a large and closely related group of environmental streptococci and streptococci-like bacteria that include bovine mastitis pathogenic Streptococcus, Enterococcus, and Aerococcus species. Phenotypic and biochemical identification methods can be inaccurate and unreliable for species within this group, particularly for Lactococcus spp. As a result, the incidence of Lactococcus spp. on the farm may have been historically underreported and consequently little is known about the clinical importance of this genus as a mastitis pathogen. We used molecular genetic identification methods to accurately differentiate 60 environmental streptococci and streptococci-like bacteria isolated from cows with high somatic cell count and chronic intramammary infection (IMI; >2 somatic cell scores above 4) among 5 geographically distinct farms in New York and Minnesota that exhibited an observed increase in IMI. These isolates were phenotypically identified as Streptococcus uberis and Streptococcus spp. Genetic methods identified 42 isolates (70%) as Lactococcus lactis ssp. lactis, including all 10 isolates originally phenotypically identified as Streptococcus uberis. Antibiotic inhibition testing of all Lc. lactis ssp. lactis showed that 7 isolates were resistant to tetracycline. In the present study, a predominance of Lc. lactis ssp. lactis was identified in association with chronic, clinical bovine IMI among all 5 farms and characterized antimicrobial resistance for treatment therapies. Routine use by mastitis testing labs of molecular identification methods for environmental streptococci and streptococci-like bacteria can further define the role and prevalence of Lc. lactis ssp. lactis in association with bovine IMI and may lead to more targeted therapies. PMID:25242419

Werner, B; Moroni, P; Gioia, G; Lavn-Alconero, L; Yousaf, A; Charter, M E; Carter, B Moslock; Bennett, J; Nydam, D V; Welcome, F; Schukken, Y H

2014-11-01

106

Effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes in continuous culture  

Microsoft Academic Search

The effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes from glucose was investigated in a microaerobic continuous culture. At a dilution rate of 0.20 h-1 and a fixed oxygen uptake rate (OUR) of 31.5 mmol l-1 h-1 the biomass concentration increased with pH ranging from 5.0 to 7.0, while the specific ATP requirement of

An-Ping Zeng; Hanno Biebl; Wolf-Dieter Deckwer

1990-01-01

107

Draft Genome Assemblies of Enterobacter aerogenes CDC 6003-71, Enterobactercloacae CDC 442-68, and Pantoea agglomerans UA 0804-01  

PubMed Central

The Enterobacteriaceae are environmental and enteric microbes. We sequenced the genomes of two Enterobacter reference strains, E.aerogenes CDC 6003-71 and E.cloacae CDC 442-68, as well as one near neighbor used as an exclusionary reference for diagnostics, Pantoea agglomerans CDC UA0804-01. The genome sizes range from 4.72 to 5.55 Mbp and have G+C contents from 54.6 to 55.1%. PMID:25342683

Minogue, T. D.; Daligault, H. E.; Davenport, K. W.; Bishop-Lilly, K. A.; Bruce, D. C.; Chain, P. S.; Coyne, S. R.; Chertkov, O.; Freitas, T.; Frey, K. G.; Jaissle, J.; Koroleva, G. I.; Ladner, J. T.; Palacios, G. F.; Redden, C. L.; Xu, Y.

2014-01-01

108

Draft Genome Assemblies of Enterobacter aerogenes CDC 6003-71, Enterobactercloacae CDC 442-68, and Pantoea agglomerans UA 0804-01.  

PubMed

The Enterobacteriaceae are environmental and enteric microbes. We sequenced the genomes of two Enterobacter reference strains, E.aerogenes CDC 6003-71 and E.cloacae CDC 442-68, as well as one near neighbor used as an exclusionary reference for diagnostics, Pantoea agglomerans CDC UA0804-01. The genome sizes range from 4.72 to 5.55 Mbp and have G+C contents from 54.6 to 55.1%. PMID:25342683

Minogue, T D; Daligault, H E; Davenport, K W; Bishop-Lilly, K A; Bruce, D C; Chain, P S; Coyne, S R; Chertkov, O; Freitas, T; Frey, K G; Jaissle, J; Koroleva, G I; Ladner, J T; Palacios, G F; Redden, C L; Xu, Y; Johnson, S L

2014-01-01

109

Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor.  

PubMed Central

A chimeric promoter with the nitrogen assimilation control protein binding site from hutUp of Klebsiella aerogenes fused to the lacZ core promoter from Escherichia coli was built and cloned in a lacZ reporter plasmid. This construct showed a 14-fold increase of beta-galactosidase activity upon nitrogen limitation. Primer extension experiments showed that the nitrogen assimilation control protein activates lacZp1 in a position-dependent manner. PMID:7642513

Pomposiello, P J; Bender, R A

1995-01-01

110

Transcriptional and translational regulation of alpha-acetolactate decarboxylase of Lactococcus lactis subsp. lactis.  

PubMed

The alpha-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in Lactococcus lactis. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of alpha-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the leu and ilv genes (P1 and P2) or independently under the control of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response. Second, although aldB is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The aldB ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure. The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the aldB RBS was responsible for a 100-fold decrease in the level of aldB translation. The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of aldB translation. Finally, the translation of the ilvA gene terminating in the latter structure decreased the level of translation of aldB fivefold more, leading to the complete extinction of the reporter gene activity. Since three leucines and one valine are present among the last six amino acids of the ilvA product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate. Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures. PMID:10986242

Goupil-Feuillerat, N; Corthier, G; Godon, J J; Ehrlich, S D; Renault, P

2000-10-01

111

Transcriptional and Translational Regulation of ?-Acetolactate Decarboxylase of Lactococcus lactis subsp. lactis  

PubMed Central

The ?-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in Lactococcus lactis. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of ?-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the leu and ilv genes (P1 and P2) or independently under the control of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response. Second, although aldB is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The aldB ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure. The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the aldB RBS was responsible for a 100-fold decrease in the level of aldB translation. The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of aldB translation. Finally, the translation of the ilvA gene terminating in the latter structure decreased the level of translation of aldB fivefold more, leading to the complete extinction of the reporter gene activity. Since three leucines and one valine are present among the last six amino acids of the ilvA product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate. Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures. PMID:10986242

Goupil-Feuillerat, Nathalie; Corthier, Grard; Godon, Jean-Jacques; Ehrlich, S. Dusko; Renault, Pierre

2000-01-01

112

Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance.  

PubMed

Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and ?-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4C); there was also a strong improvement in cell survival in response to heat shock (45C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery. PMID:21515730

Carvalho, Ana Lcia; Cardoso, Filipa S; Bohn, Andreas; Neves, Ana Rute; Santos, Helena

2011-06-01

113

Integrated evaluation of aerogenic pollution by air-transported heavy metals (Pb, Cd, Ni, Zn, Mn and Cu) in the analysis of the main deposit media.  

PubMed

The composition of the ambient air is constantly changing; therefore, the monitoring of ambient air quality to detect the changes caused by aerogenic pollutants makes the essential part of general environmental monitoring. To achieve more effective improvement of the ambient air quality, the Directive 2008/50/EC on 'Ambient Air Quality and Cleaner Air for Europe' was adopted by the European Parliament and the European Council. It informed the public and enterprises about a negative effect of pollution on humans, animals and plants, as well as about the need for monitoring aerogenic pollutants not only at the continuous monitoring stations but also by using indicator methods, i.e. by analysing natural deposit media. The problem of determining the relationship between the accumulation level of pollutants by a deposit medium and the level of air pollution and its risks is constantly growing in importance. The paper presents a comprehensive analysis of the response of the main four deposit media, i.e. snow cover, soil, pine bark and epigeic mosses, to the long-term pollution by aerogenic pollutants which can be observed in the area of oil refinery influence. Based on the quantitative expressions of the amounts of the accumulated pollutants in the deposit media, the territory of the oil refinery investigated in this paper has been referred to the areas of mild or moderate pollution. PMID:23933956

Baltr?nait?, Edita; Baltr?nas, Pranas; Lietuvninkas, Arvydas; Serevi?ien?, Vaida; Zuokait?, Egl?

2014-01-01

114

Functional expression of plant membrane proteins in Lactococcus lactis.  

PubMed

The study of most membrane proteins remains challenging due to their hydrophobicity and their low natural abundance in cells. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations and is nowadays widely used in biotechnology for large-scale production of heterologous proteins. This system has been successfully used for the production of prokaryotic and eukaryotic membrane proteins. The purpose of this chapter is to provide detailed protocols for (1) the expression of plant peripheral or intrinsic membrane proteins and then for (2) their solubilization, from Lactococcus membranes, for further purification steps and biochemical characterization. PMID:25447863

Boutigny, Sylvain; Sautron, Emeline; Frelet-Barrand, Annie; Moyet, Lucas; Salvi, Daniel; Rolland, Norbert; Seigneurin-Berny, Daphn

2015-01-01

115

Sequencing and transcriptional analysis of the biosynthesis gene cluster of putrescine-producing Lactococcus lactis.  

PubMed

Lactococcus lactis is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of L. lactis in dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains of L. lactis subsp. lactis and L. lactis subsp. cremoris that are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster of L. lactis is composed of a putative regulatory gene, aguR, followed by the genes (aguB, aguD, aguA, and aguC) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing L. lactis strains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution. PMID:21803900

Ladero, Victor; Rattray, Fergal P; Mayo, Baltasar; Martn, Mara Cruz; Fernndez, Mara; Alvarez, Miguel A

2011-09-01

116

Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis ?  

PubMed Central

Lactococcus lactis is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of L. lactis in dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains of L. lactis subsp. lactis and L. lactis subsp. cremoris that are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster of L. lactis is composed of a putative regulatory gene, aguR, followed by the genes (aguB, aguD, aguA, and aguC) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing L. lactis strains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution. PMID:21803900

Ladero, Victor; Rattray, Fergal P.; Mayo, Baltasar; Martn, Mara Cruz; Fernndez, Mara; Alvarez, Miguel A.

2011-01-01

117

Diversity Analysis of Dairy and Nondairy Lactococcus lactis Isolates, Using a Novel Multilocus Sequence Analysis Scheme and (GTG)5-PCR Fingerprinting?  

PubMed Central

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)5-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene. PMID:17890345

Rademaker, Jan L. W.; Herbet, Hlne; Starrenburg, Marjo J. C.; Naser, Sabri M.; Gevers, Dirk; Kelly, William J.; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E. T.

2007-01-01

118

Cholate-Stimulated Biofilm Formation by Lactococcus lactis Cells ?  

PubMed Central

Bile acid resistance by Lactococcus lactis depends on the ABC-type multidrug transporter LmrCD. Upon deletion of the lmrCD genes, cells can reacquire bile acid resistance upon prolonged exposure to cholate, yielding the ?lmrCDr strain. The resistance mechanism in this strain is non-transporter based. Instead, cells show a high tendency to flocculate, suggesting cell surface alterations. Contact angle measurements demonstrate that the ?lmrCDr cells are equipped with an increased cell surface hydrophilicity compared to those of the parental and wild-type strains, while the surface hydrophilicity is reduced in the presence of cholate. ?lmrCDr cells are poor in biofilm formation on a hydrophobic polystyrene surface, but in the presence of subinhibitory concentrations of cholate, biofilm formation is strongly stimulated. Biofilm cells show an enhanced extracellular polymeric substance production and are highly resistant to bile acids. These data suggest that non-transporter-based cholate resistance in L. lactis is due to alterations in the cell surface that stimulate cells to form resistant biofilms. PMID:21335382

Zaidi, Arsalan Haseeb; Bakkes, Patrick J.; Krom, Bastiaan P.; van der Mei, Henny C.; Driessen, Arnold J. M.

2011-01-01

119

Transcriptional analysis of oligosaccharide utilization by Bifidobacterium lactis Bl-04  

PubMed Central

Background Probiotic bifidobacteria in combination with prebiotic carbohydrates have documented positive effects on human health regarding gastrointestinal disorders and improved immunity, however the selective routes of uptake remain unknown for most candidate prebiotics. The differential transcriptomes of Bifidobacterium animalis subsp. lactis Bl-04, induced by 11 potential prebiotic oligosaccharides were analyzed to identify the genetic loci involved in the uptake and catabolism of ?- and ?-linked hexoses, and ?-xylosides. Results The overall transcriptome was modulated dependent on the type of glycoside (galactosides, glucosides or xylosides) utilized. Carbohydrate transporters of the major facilitator superfamily (induced by gentiobiose and ?-galacto-oligosaccharides (GOS)) and ATP-binding cassette (ABC) transporters (upregulated by cellobiose, GOS, isomaltose, maltotriose, melibiose, panose, raffinose, stachyose, xylobiose and ?-xylo-oligosaccharides) were differentially upregulated, together with glycoside hydrolases from families 1, 2, 13, 36, 42, 43 and 77. Sequence analysis of the identified solute-binding proteins that determine the specificity of ABC transporters revealed similarities in the breadth and selectivity of prebiotic utilization by bifidobacteria. Conclusion This study identified the differential gene expression for utilization of potential prebiotics highlighting the extensive capabilities of Bifidobacterium lactis Bl-04 to utilize oligosaccharides. Results provide insights into the ability of this probiotic microbe to utilize indigestible carbohydrates in the human gastrointestinal tract. PMID:23663691

2013-01-01

120

Repression of Glutamate Dehydrogenase Formation in Klebsiella aerogenes Requires Two Binding Sites for the Nitrogen Assimilation Control Protein, NAC  

PubMed Central

In Klebsiella aerogenes, the gdhA gene codes for glutamate dehydrogenase, one of the enzymes responsible for assimilating ammonia into glutamate. Expression of a gdhAp-lacZ transcriptional fusion was strongly repressed by the nitrogen assimilation control protein, NAC. This strong repression (>50-fold under conditions of severe nitrogen limitation) required the presence of two separate NAC binding sites centered at ?89 and +57 relative to the start of gdhA transcription. Mutants lacking either or both of these sites lost the strong repression. The distance between the two sites was less important than the face of the helix on which they lay. Insertion or deletion of 10 bp between the sites had little effect on the strong repression, but insertion of 5 bp or deletion of either 5 or 15 bp decreased the repression significantly. We propose that the strong repression of gdhAp-lacZ expression requires an interaction between the NAC molecules bound at the two sites. A weaker repression of gdhAp-lacZ expression (about threefold) required only the NAC site centered at ?89. This weaker repression appears to result from NAC's ability to prevent the action of a positive effector the target of which overlaps the NAC binding site centered at ?89. Point mutations and deletions of this region result in the same threefold reduction in gdhAp-lacZ expression as the presence of NAC at this site. PMID:12446647

Goss, Thomas J.; Janes, Brian K.; Bender, Robert A.

2002-01-01

121

An evaluation and partial characterization of a bacteriocin produced by Lactococcus lactis subsp lactis ST1 isolated from goat milk  

PubMed Central

A bacteriocin-like inhibitory substance producing Lactococcus lactis subsp lactis strain, ST1, isolated from goat milk of Iranian origin and with broad spectrum of activity and desirable technical properties was used for evaluating some futures of bacteriocin inhibitory activity. Cell growth and bacteriocin production studies were carried out in MRS medium incubated statically under uncontrolled pH condition. The antibacterial activity presented a primary metabolite pattern and showed a rapid decrease at the stationary phase. Microaerobiosis and capnophily growth conditions resulted in higher bacteriocin production while aerobiosis showed negative effect on both cell growth and bacteriocin production. Bacteriocin production, on the other hand, was favored in MRS broth (pH; 6.5) inoculated with 0.1 ml l-1 fresh culture when incubation was carried out at 30 C. This indicated that the conditions resulted in higher levels of growth were frequently favoring bacteriocin production by ST1 as well. Decrease in activity, at the stationary growth phase, was much pronounced in favored growth condition. Nutrient depletion, deferent effect of low pH on bacteriocin production and/or protein degradation seemed more responsible for this phenomenon. The study also provided further data on new method for bacteriocin release from the cell wall of producer. It was clearly shown that both heating and ultrasound shock for 5 min at pH 2 could increase bacteriocin activity significantly. The release was more pronounced in the presence of 0.5% Tween80. PMID:24031976

Taheri, Parinaz; Samadi, Nasrin; Ehsani, Mohammad Reza; Khoshayand, Mohammad Reza; Jamalifar, Hossein

2012-01-01

122

Study of the Citrate Metabolism of Lactococcus lactis subsp. lactis Biovar Diacetylactis by Means of 13C Nuclear Magnetic Resonance  

PubMed Central

The metabolic fate of citrate and pyruvate in four strains of Lactococcus lactis subsp. lactis biovar diacetylactis has been studied by means of 13C nuclear magnetic resonance, using as a substrate either [3-13C]pyruvic acid or custom-synthesized citric acid that is 13C labeled either at carbons 2 and 4 or at carbon 3. The fermentations were carried out batchwise in modified M17 broth. For the actual conversions of the 13C-labeled substrates, cells at the end of their logarithmic growth phase were used to minimize the conversion to lactic acid. A mass balance of the main citric acid metabolites was obtained; the four strains produced from 50 to 70% (on a molar basis) lactic acid from either citrate or pyruvate. The remaining 50 to 30% was converted mainly to either ?-acetolactic acid (for one strain) or acetoin (for the other three strains). One of the strains produced an exceptionally high concentration of the diacetyl precursor ?-acetolactic acid. Another strain (SDC6) also produced ?-acetolactic acid, but this was decarboxylated to acetoin at a high rate. The 13C nuclear magnetic resonance method confirmed that the biosynthesis of ?-acetolactic acid occurs via condensation of pyruvate and active acetaldehyde. Diacetyl was not found as a direct metabolite of citrate or pyruvate metabolism. PMID:16348592

Verhue, Walter M.; Tjan, Frans S. B.

1991-01-01

123

Riboflavin production in Lactococcus lactis: potential for in situ production of vitamin-enriched foods.  

PubMed

This study describes the genetic analysis of the riboflavin (vitamin B(2)) 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. PMID:15466513

Burgess, Catherine; O'connell-Motherway, Mary; Sybesma, Wilbert; Hugenholtz, Jeroen; van Sinderen, Douwe

2004-10-01

124

21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.  

Code of Federal Regulations, 2010 CFR

...2009-04-01 true Aminopeptidase enzyme preparation derived from lactococcus...GRAS 184.1985 Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation is derived from the...

2010-04-01

125

21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.  

Code of Federal Regulations, 2013 CFR

...2013-04-01 false Aminopeptidase enzyme preparation derived from lactococcus...GRAS 184.1985 Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation is derived from the...

2013-04-01

126

21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.  

Code of Federal Regulations, 2014 CFR

...2014-04-01 false Aminopeptidase enzyme preparation derived from lactococcus...GRAS 184.1985 Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation is derived from the...

2014-04-01

127

21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.  

Code of Federal Regulations, 2012 CFR

...2012-04-01 false Aminopeptidase enzyme preparation derived from lactococcus...GRAS 184.1985 Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation is derived from the...

2012-04-01

128

21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.  

Code of Federal Regulations, 2011 CFR

...2011-04-01 false Aminopeptidase enzyme preparation derived from lactococcus...GRAS 184.1985 Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation is derived from the...

2011-04-01

129

Two mechanisms for oxidation of cytosolic NADPH by Kluyveromyces lactis mitochondria.  

PubMed

Null mutations in the structural gene encoding phosphoglucose isomerase completely abolish activity of this glycolytic enzyme in Kluyveromyces lactis and Saccharomyces cerevisiae. In S. cerevisiae, the pgi1 null mutation abolishes growth on glucose, whereas K.lactis rag2 null mutants still grow on glucose. It has been proposed that, in the latter case, growth on glucose is made possible by an ability of K. lactis mitochondria to oxidize cytosolic NADPH. This would allow for a re-routing of glucose dissimilation via the pentose-phosphate pathway. Consistent with this hypothesis, mitochondria of S. cerevisiae cannot oxidize NADPH. In the present study, the ability of K. lactis mitochondria to oxidize cytosolic NADPH was experimentally investigated. Respiration-competent mitochondria were isolated from aerobic, glucose-limited chemostat cultures of the wild-type K. lactis strain CBS 2359 and from an isogenic rag2Delta strain. Oxygen-uptake experiments confirmed the presence of a mitochondrial NADPH dehydrogenase in K.lactis. This activity was ca. 2.5-fold higher in the rag2Delta mutant than in the wild-type strain. In contrast to mitochondria from wild-type K. lactis, mitochondria from the rag2Delta mutant exhibited high rates of ethanol-dependent oxygen uptake. Subcellular fractionation studies demonstrated that, in the rag2Delta mutant, a mitochondrial alcohol dehydrogenase was present and that activity of a cytosolic NADPH-dependent 'acetaldehyde reductase' was also increased. These observations indicate that two mechanisms may participate in mitochondrial oxidation of cytosolic NADPH by K. lactis mitochondria: (a) direct oxidation of cytosolic NADPH by a mitochondrial NADPH dehydrogenase; and (b) a two-compartment transhydrogenase cycle involving NADP(+)- and NAD(+)-dependent alcohol dehydrogenases. PMID:12112236

Overkamp, Karin M; Bakker, Barbara M; Steensma, H Y; van Dijken, Johannes P; Pronk, Jack T

2002-07-01

130

Lactococcus lactis Metabolism and Gene Expression during Growth on Plant Tissues.  

PubMed

Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits of Lactococcus lactis during growth in an Arabidopsis thaliana leaf tissue lysate (ATL). L. lactis KF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strain L. lactis IL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded with L. lactis KF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes for L. lactis growth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-type L. lactis strain KF147 but not an xylA deletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. lactis carbohydrate metabolism on plant tissues. These findings show that certain strains of L. lactis are well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations. PMID:25384484

Golomb, Benjamin L; Marco, Maria L

2015-01-15

131

Oral immunization with recombinant Lactococcus lactis confers protection against respiratory pneumococcal infection.  

PubMed

In the present work, we evaluated if oral immunization with the pneumococcal protective protein A (PppA), expressed in the cell wall of Lactococcus lactis (L. lactis PppA+), was able to confer protective immunity against Streptococcus pneumoniae. Mice were immunized orally with L. lactis PppA+ for 5 consecutive days. Vaccination was performed one (nonboosted group) or 2 times with a 2 week interval between each immunization (boosted group). Oral priming with L. lactis PppA+ induced the production of anti-PppA IgM, IgG, and IgA antibodies in serum and in bronchoalveolar (BAL) and intestinal (IF) lavage fluids. Boosting with L. lactis PppA+ increased the levels of mucosal and systemic immunoglobulins. Moreover, the avidity and the opsonophagocytic activity of anti-PppA antibodies were significantly improved in the boosted group. The presence of both IgG1 and IgG2a anti-PppA antibodies in serum and BAL and the production of both interferon gamma and interleukin-4 by spleen cells from immunized mice indicated that L. lactis PppA+ stimulated a mixture of Th1 and Th2 responses. The ability of L. lactis PppA+ to confer cross-protective immunity was evaluated using challenge assays with serotypes 3, 6B, 14, and 23F. Lung bacterial cell counts and hemocultures showed that immunization with L. lactis PppA+ improved resistance against all the serotypes assessed, including serotype 3, which was highly virulent in our experimental animal model. To our knowledge, this is the first demonstration of protection against respiratory pneumococcal infection induced by oral administration of a recombinant lactococcal vaccine. PMID:18923553

Villena, Julio; Medina, Marcela; Raya, Ral; Alvarez, Susana

2008-10-01

132

Morphology, Genome Sequence, and Structural Proteome of Type Phage P335 from Lactococcus lactis  

Microsoft Academic Search

Lactococcus lactis phage P335 is a virulent type phage for the species that bears its name and belongs to the Siphoviridae family. Morphologically, P335 resembled the L. lactis phages TP901-1 and Tuc2009, except for a shorter tail and a different collar\\/whisker structure. Its 33,613-bp double-stranded DNA genome had 50 open reading frames. Putative functions were assigned to 29 of them.

Simon J. Labrie; Jytte Josephsen; Horst Neve; Finn K. Vogensen; Sylvain Moineau

2008-01-01

133

Generation of a membrane potential by Lactococcus lactis through aerobic electron transport  

Microsoft Academic Search

Lactococcus lactis, a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in L. lactis under these conditions. Proton motive force generation in whole cells was measured using a fluorescent probe (3',3'-dipropylthiadicarbocyanine),

R. J. W. Brooijmans; B. Poolman; G. K. Schuurman-Wolters; W. M. de Vos; J. Hugenholtz

2007-01-01

134

Peptidase and proteinase activity of Lactococcus lactis, Lactobacillus casei and Lactobacillus plantarum  

Microsoft Academic Search

Zusammenfassung Das proteolytische System von mehreren nicht kommerziellenLactococcus- undLactobacillus-Stmmen wurde direkt vom traditionellen spanischen halbfesten Ziegenmilchkse isoliert und untersucht. Die Aktivitt von Aminopeptidase, X-Prolyldipeptidylaminopeptidase, Dipeptidase and Proteinase dieser neuen Stmme wurde in cytoplasmatischen, Zellwand-Membran- und spontan freigesetzten Fraktionen gemessen. Die Aminopeptidase-Aktivitt erfolgte ausschlielich intracellular und war hher frLactobacillus casei subsp. casei als frLactococcus lactis subsp.lactis. Lactobacillus plantarum zeigte hhere Dipeptidase

Teresa Requena; Carmen Pelaez; Patrick F. Fox

1993-01-01

135

Nasal immunization with Lactococcus lactis expressing the pneumococcal protective protein A induces protective immunity in mice.  

PubMed

Nisin-controlled gene expression was used to develop a recombinant strain of Lactococcus lactis that is able to express the pneumococcal protective protein A (PppA) on its surface. Immunodetection assays confirmed that after the induction with nisin, the PppA antigen was predictably and efficiently displayed on the cell surface of the recombinant strain, which was termed L. lactis PppA. The production of mucosal and systemically specific antibodies in adult and young mice was evaluated after mice were nasally immunized with L. lactis PppA. Immunoglobulin M (IgM), IgG, and IgA anti-PppA antibodies were detected in the serum and bronchoalveolar lavage fluid of adult and young mice, which showed that PppA expressed in L. lactis was able to induce a strong mucosal and systemic immune response. Challenge survival experiments demonstrated that immunization with L. lactis PppA was able to increase resistance to systemic and respiratory infection with different pneumococcal serotypes, and passive immunization assays of nave young mice demonstrated a direct correlation between anti-PppA antibodies and protection. The results presented in this study demonstrate three major characteristics of the effectiveness of nasal immunization with PppA expressed as a protein anchored to the cell wall of L. lactis: it elicited cross-protective immunity against different pneumococcal serotypes, it afforded protection against both systemic and respiratory challenges, and it induced protective immunity in mice of different ages. PMID:18390997

Medina, Marcela; Villena, Julio; Vintii, Elisa; Hebert, Elvira Mara; Raya, Ral; Alvarez, Susana

2008-06-01

136

Effects of metal ions on growth, ?-oxidation system, and thioesterase activity of Lactococcus lactis.  

PubMed

The effects of divalent metal ions (Ca(2+), Mg(2+), Fe(2+), and Cu(2+)) on the growth, ?-oxidation system, and thioesterase activity of Lactococcus lactis were investigated. Different metal ions significantly influenced the growth of L. lactis: Ca(2+) and Fe(2+) accelerated growth, whereas Cu(2+) inhibited growth. Furthermore, Mg(2+) inhibited growth of L. lactis at a low concentration but stimulated growth of L. lactis at a high concentration. The divalent metal ions had significant effects on activity of the 4 key enzymes of the ?-oxidation system (acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and thiolase) and thioesterase of L. lactis. The activity of acyl-CoA dehydrogenases increased markedly in the presence of Ca(2+) and Mg(2+), whereas it decreased with 1 mmol/L Fe(2+) or 12 mmol/L Mg(2+). All the metal ions could induce activity of enoyl-CoA hydratase. In addition, 12 mmol/L Mg(2+) significantly stimulated activity of L-3-hydroxyacyl-CoA dehydrogenase, and all metal ions could induce activity of thiolase, although thiolase activity decreased significantly when 0.05 mmol/L Cu(2+) was added into M17 broth. Inhibition of thioesterase activity by all 4 metal ions could be reversed by 2 mmol/L Ca(2+). These results help us understand the effect of metal ions on the ?-oxidation system and thioesterase activity of Lactococcus lactis. PMID:25064652

Li, Liang; Ma, Ying

2014-10-01

137

Isolation and characterization of a pur?C(or?f)QLF operon from Lactobacillus lactis MG1614  

Microsoft Academic Search

We have isolated genes encoding enzymes of the de novo purine nucleotide biosynthesis pathway from Lactococcus lactis MG1614 by colony hybridization using DIG-labeled DNA probes. The organization of the genes needed for the de novo biosynthesis\\u000a of purine nucleotides in L. lactis differs from that found in other organisms. In L. lactis there is a gene cluster, which contains five

T. Peltonen; P. Mntsl

1999-01-01

138

Carboxylic acids permeases in yeast: two genes in Kluyveromyces lactis.  

PubMed

Two new genes KlJEN1 and KlJEN2 were identified in Kluyveromyces lactis. The deduced structure of their products is typical of membrane-bound carriers and displays high similarity to Jen1p, the monocarboxylate permease of Saccharomyces cerevisiae. Both KlJEN1 and KlJEN2 are under the control of glucose repression mediated by FOG1 and FOG2, corresponding to S. cerevisiae GAL83 and SNF1 respectively, and KlCAT8, proteins involved in glucose signalling cascade in K. lactis. KlJEN1, but not KlJEN2, is induced by lactate. KlJEN2 in contrast is expressed at high level in ethanol and succinate. The physiological characterization of null mutants showed that KlJEN1 is the functional homologue of ScJEN1, whereas KlJEN2 encodes a dicarboxylic acids transporter. In fact, KlJen1p [transporter classification (TC) number: 2.A.1.12.2.] is required for lactate uptake and therefore for growth on lactate. KlJen2p is required for succinate transport, as demonstrated by succinate uptake experiments and by inability of Kljen2 mutant to grow on succinate. This carrier appears to transport also malate and fumarate because the Kljen2 mutant cannot grow on these substrates and the succinate uptake is competed by these carboxylic acids. We conclude that KlJEN2 is the first yeast gene shown to encode a dicarboxylic acids permease. PMID:15363851

Lodi, Tiziana; Fontanesi, Flavia; Ferrero, Iliana; Donnini, Claudia

2004-09-15

139

Regulatory Functions of Serine-46-Phosphorylated HPr in Lactococcus lactis  

PubMed Central

In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a ?-glucoside-specific EII and a 6-P-?-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methyl ?-d-thiogalactoside (TMG) and 2-deoxy-d-glucose (2-DG). In vivo experiments with the ptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process. PMID:11344147

Monedero, Vicente; Kuipers, Oscar P.; Jamet, Emmanuel; Deutscher, Josef

2001-01-01

140

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes.  

PubMed

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ?adhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ?adhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production. PMID:25416770

Tajima, Yoshinori; Yamamoto, Yoko; Fukui, Keita; Nishio, Yousuke; Hashiguchi, Kenichi; Usuda, Yoshihiro; Sode, Koji

2015-02-01

141

Alanine Catabolism in Klebsiella aerogenes: Molecular Characterization of the dadAB Operon and Its Regulation by the Nitrogen Assimilation Control Protein  

PubMed Central

Klebsiella aerogenes strains with reduced levels of d-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of d-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position ?44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein. PMID:9457858

Janes, Brian K.; Bender, Robert A.

1998-01-01

142

Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities  

SciTech Connect

A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of (/sup 14/C)lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution /sup 31/P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM.

Thompson, J.; Chassy, B.M.; Egan, W.

1985-04-01

143

Interaction between the genomes of Lactococcus lactis and phages of the P335 species  

PubMed Central

Phages of the P335 species infect Lactococcus lactis and have been particularly studied because of their association with strains of L. lactis subsp. cremoris used as dairy starter cultures. Unlike other lactococcal phages, those of the P335 species may have a temperate or lytic lifestyle, and are believed to originate from the starter cultures themselves. We have sequenced the genome of L. lactis subsp. cremoris KW2 isolated from fermented corn and found that it contains an integrated P335 species prophage. This 41 kb prophage (? KW2) has a mosaic structure with functional modules that are highly similar to several other phages of the P335 species associated with dairy starter cultures. Comparison of the genomes of 26 phages of the P335 species, with either a lytic or temperate lifestyle, shows that they can be divided into three groups and that the morphogenesis gene region is the most conserved. Analysis of these phage genomes in conjunction with the genomes of several L. lactis strains shows that prophage insertion is site specific and occurs at seven different chromosomal locations. Exactly how induced or lytic phages of the P335 species interact with carbohydrate cell surface receptors in the host cell envelope remains to be determined. Genes for the biosynthesis of a variable cell surface polysaccharide and for lipoteichoic acids (LTAs) are found in L. lactis and are the main candidates for phage receptors, as the genes for other cell surface carbohydrates have been lost from dairy starter strains. Overall, phages of the P335 species appear to have had only a minor role in the adaptation of L. lactis subsp. cremoris strains to the dairy environment, and instead they appear to be an integral part of the L. lactis chromosome. There remains a great deal to be discovered about their role, and their contribution to the evolution of the bacterial genome. PMID:24009606

Kelly, William J.; Altermann, Eric; Lambie, Suzanne C.; Leahy, Sinead C.

2013-01-01

144

Interaction between the genomes of Lactococcus lactis and phages of the P335 species.  

PubMed

Phages of the P335 species infect Lactococcus lactis and have been particularly studied because of their association with strains of L. lactis subsp. cremoris used as dairy starter cultures. Unlike other lactococcal phages, those of the P335 species may have a temperate or lytic lifestyle, and are believed to originate from the starter cultures themselves. We have sequenced the genome of L. lactis subsp. cremoris KW2 isolated from fermented corn and found that it contains an integrated P335 species prophage. This 41 kb prophage (? KW2) has a mosaic structure with functional modules that are highly similar to several other phages of the P335 species associated with dairy starter cultures. Comparison of the genomes of 26 phages of the P335 species, with either a lytic or temperate lifestyle, shows that they can be divided into three groups and that the morphogenesis gene region is the most conserved. Analysis of these phage genomes in conjunction with the genomes of several L. lactis strains shows that prophage insertion is site specific and occurs at seven different chromosomal locations. Exactly how induced or lytic phages of the P335 species interact with carbohydrate cell surface receptors in the host cell envelope remains to be determined. Genes for the biosynthesis of a variable cell surface polysaccharide and for lipoteichoic acids (LTAs) are found in L. lactis and are the main candidates for phage receptors, as the genes for other cell surface carbohydrates have been lost from dairy starter strains. Overall, phages of the P335 species appear to have had only a minor role in the adaptation of L. lactis subsp. cremoris strains to the dairy environment, and instead they appear to be an integral part of the L. lactis chromosome. There remains a great deal to be discovered about their role, and their contribution to the evolution of the bacterial genome. PMID:24009606

Kelly, William J; Altermann, Eric; Lambie, Suzanne C; Leahy, Sinead C

2013-01-01

145

A comparative study on phosphotransferase activity of acid phosphatases from Raoultella planticola and Enterobacter aerogenes on nucleosides, sugars, and related compounds.  

PubMed

Natural and modified nucleoside-5'-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained. PMID:23995227

Mdici, Rosario; Garaycoechea, Juan I; Valino, Ana L; Pereira, Claudio A; Lewkowicz, Elizabeth S; Iribarren, Adolfo M

2014-04-01

146

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation.  

PubMed Central

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms. Images PMID:1901708

Broadbent, J R; Kondo, J K

1991-01-01

147

Multi-stress resistance in Lactococcus lactis is actually escape from purine-induced stress sensitivity.  

PubMed

Multi-stress resistance is a widely documented and fascinating phenotype of lactococci where single mutations, preferentially in genes involved in nucleotide metabolism and phosphate uptake, result in elevated tolerance to multiple stresses simultaneously. In this report, we have analysed the metabolic basis behind this multi-stress-resistance phenotype in Lactococcus lactis subsp. cremoris MG1363 using acid stress as a model of multi-stress resistance. Surprisingly, we found that L. lactis MG1363 is fully resistant to pH 3.0 in the chemically defined SA medium, contrary to its sensitivity in the rich and complex M17 medium. When salvage of purines and subsequent conversion to GTP was permitted in various genetic backgrounds of L. lactis MG1363, the cells became sensitive to acid stress, indicating that an excess of guanine nucleotides induces stress sensitivity. The addition of phosphate to the acid-stress medium increased the stress sensitivity of L. lactis MG1363. It is also shown that high intracellular guanine nucleotide pools confer increased sensitivity to high temperatures, thus showing that it is indeed a multi-stress phenotype. Our analysis suggests that an increased level of guanine nucleotides is formed as a result of an improved conversion of guanosine in the salvage pathway. Based upon our findings, we suggest that L. lactis MG1363 is naturally multi-stress resistant in habitats devoid of any purine source. However, any exogenous purine that results in increased guanine nucleotide pools renders the bacterium sensitive to environmental stresses. PMID:25143058

Ryssel, Mia; Hviid, Anne-Mette Meisner; Dawish, Mohamed S; Haaber, Jakob; Hammer, Karin; Martinussen, Jan; Kilstrup, Mogens

2014-11-01

148

Transcriptomic Response of Lactococcus lactis in Mixed Culture with Staphylococcus aureus?  

PubMed Central

The mechanisms of interaction between Lactococcus lactis and the food pathogen Staphylococcus aureus are of crucial importance, as one major role of lactic acid bacteria (LAB) in fermented foods is to inhibit undesirable and pathogenic flora. It was never questioned if the presence of a pathogen can actively modify the gene expression patterns of LAB in a shared environment. In this study, transcriptome and biochemical analyses were combined to assess the dynamic response of L. lactis in a mixed culture with S. aureus. The presence of S. aureus hardly affected the growth of L. lactis but dramatically modified its gene expression profile. The main effect was related to earlier carbon limitation and a concomitantly lower growth rate in the mixed culture due to the consumption of glucose by both species. More specific responses involved diverse cellular functions. Genes associated with amino acid metabolism, ion transport, oxygen response, menaquinone metabolism, and cell surface and phage expression were differentially expressed in the mixed culture. This study led to new insights into possible mechanisms of interaction between L. lactis and S. aureus. Moreover, new and unexpected effects of L. lactis on the virulence of S. aureus were discovered, as described elsewhere (S. Even, C. Charlier, S. Nouaille, N. L. Ben Zakour, M. Cretenet, F. J. Cousin, M. Gautier, M. Cocaign-Bousquet, P. Loubire, and Y. Le Loir, Appl. Environ. Microbiol. 75:4459-4472, 2009). PMID:19429566

Nouaille, Sbastien; Even, Sergine; Charlier, Cathy; Le Loir, Yves; Cocaign-Bousquet, Muriel; Loubire, Pascal

2009-01-01

149

Intranasal immunization of recombinant Lactococcus lactis induces protection against H5N1 virus in ferrets.  

PubMed

The increasing outbreaks of highly pathogenic avian influenza A (HPAI) H5N1 viruses in birds and human bring out an urgent need to develop a safe and effective vaccine to control and prevent H5N1 infection. Lactococcus lactis (L. lactis) based vaccine platform is a promising approach for mucosal H5N1 vaccine development. Intranasal immunization is the potential to induce mucosal immune response which is associated with protective immunity. To develop a safe and effective mucosal vaccine against HAPI H5N1, we extended our previous study by evaluating the immunogenicity of L. lactis-psA-HA1 in the absence of adjuvant via intranasal route in the ferret model. Ferrets administered intranasally with L. lactis-pgsA-HA1 could elicit robust humoral and mucosal immune responses, as well as significant HI titers. Importantly, ferrets were completely protected from H5N1 virus challenge. These findings suggest that L. lactis-pgsA-HA1 can be considered an alternative mucosal vaccine during A/H5N1 pandemic. PMID:25445345

Lei, Han; Peng, Xiaojue; Ouyang, Jiexiu; Zhao, Daxian; Jiao, Huifeng; Shu, Handing; Ge, Xinqi

2015-01-22

150

Geraniol dehydrogenase, the key enzyme in biosynthesis of the alarm pheromone, from the astigmatid mite Carpoglyphus lactis (Acari: Carpoglyphidae).  

PubMed

Geraniol dehydrogenase (GeDH), which plays an important role in the biosynthesis of neral, an alarm pheromone, was purified from the astigmatid mite Carpoglyphus lactis. The enzyme was obtained in an apparently homogeneous and active form after 1879-fold purification through seven steps of chromatography. Car. lactis GeDH was determined to be a monomer in its active form with a relative molecular mass of 42 800, which is a unique subunit structure in comparison with already established alcohol dehydrogenases. Car. lactis GeDH oxidized geraniol into geranial in the presence of NAD+. NADP+ was ineffective as a cofactor, suggesting that Car. lactis GeDH is an NAD+-dependent alcohol dehydrogenase. The optimal pH and temperature for geraniol oxidation were determined to be pH 9.0 and 25 degrees C, respectively. The Km values for geraniol and NAD+ were 51.0 microm and 59.5 microm, respectively. Car. lactis GeDH was shown to selectively oxidize geraniol, whereas its geometrical isomer, nerol, was inert as a substrate. The high specificity for geraniol suggests that Car. lactis GeDH specializes in the alarm pheromone biosynthesis of Car. lactis. Car. lactis GeDH is composed of 378 amino acids. Structurally, Car. lactis GeDH showed homology with zinc-dependent alcohol dehydrogenases found in mammals and a mosquito (36.6-37.6% identical), and the enzyme was considered to be a member of the medium-chain dehydrogenase/reductase family, in view of the highly conserved sequences of zinc-binding and NAD+-binding sites. Phylogenetic analyses indicate that Car. lactis GeDH could be categorized as a new class, different from other established alcohol dehydrogenases. PMID:18422649

Noge, Koji; Kato, Makiko; Mori, Naoki; Kataoka, Michihiko; Tanaka, Chihiro; Yamasue, Yuji; Nishida, Ritsuo; Kuwahara, Yasumasa

2008-06-01

151

Spermatogenesis and sperm structure in Carpoglyphus lactis (L.) (Acari: Astigmata).  

PubMed

Testes, spermatogenesis and spermatozoa are described in the mite Carpoglyphus lactis (L.), the first representative of the Hemisarcoptoidea superfamily studied ultrastructurally. Paired testes are located posteriorly in the idiosoma, with germaria situated dorsolaterally. The germarium consists of a compact group of spermatogonia; no testicular central cell was found. The remainder of the gonad is occupied by germ cells in different stages of spermatogenesis, distributed separately rather than in cysts, and embedded in a few large somatic cells filling the remaining space. Spermatocytes are covered by a spongy layer, a product of the Golgi apparatus. Spermatids are anucleate. Their chromatin condenses into granular and then tubular threads. As spermiogenesis progresses, the spongy layer assembles at a single site and forms a structure termed the spongy body; mitochondria become electron dense, elongate and gather forming a bundle; a narrow ER cistern, promptly transforming into a dense lamella, appears between the mitochondria and chromatin. Mature spermatozoa are small, highly electron-dense cells interdigitating with others via superficial protrusions. They possess chromatin threads, electron-dense lamella and mitochondria, but do not have an acrosome. Our results support the monophyly of Astigmata, but do not explain the phylogenetic affinities of Hemisarcoptoidea to other superfamilies of astigmatic mites. PMID:19766733

Florek, Maria; Witali?ski, Wojciech

2010-01-01

152

Continuous nisin production with bioengineered Lactococcus lactis strains.  

PubMed

Nisin production in continuous cultures of bioengineered Lactococcus lactis strains that incorporate additional immunity and regulation genes was studied. Highest nisin activities were observed at 0.2 h(-1) dilution rate and 12.5 g l(-1) fructose concentration for all strains. Recombinant strains were able to produce greater amounts of nisin at dilution rates below 0.3 h(-1) compared to the control strain. However, this significant difference disappeared at dilution rates of 0.4 and 0.5 h(-1). For the strains LL27, LAC338, LAC339, and LAC340, optimum conditions for nisin production were determined to be at 0.29, 0.26, 0.27, and 0.27 h(-1) dilution rates and 11.95, 12.01, 11.63, and 12.50 g l(-1) fructose concentrations, respectively. The highest nisin productivity, 496 IU ml(-1) h(-1), was achieved with LAC339. The results of this study suggest that low dilution rates stabilize the high specific nisin productivity of the bioengineered strains in continuous fermentation. Moreover, response surface methodology analysis showed that regulation genes yielded high nisin productivity at wide ranges of dilution rates and fructose concentrations. PMID:19337764

Sim?ek, O; Akko, N; Con, A H; Ozelik, F; Saris, P E J; Akelik, Mustafa

2009-06-01

153

[The humoral immune response in mice induced by recombinant Lactococcus lactis expressing HIV-1 gag].  

PubMed

Objective To analyze the humoral immune response induced by recombinant Lactococcus lactis expressing HIV-1 gag in mice immunized orally, intranasally, subcutaneously or in the combined way of above three. Methods Fifty BALB/c mice were randomly divided into 5 groups, 10 mice per group. The mice were immunized consecutively three times at two week intervals with 10(9) CFU of recombinant Lactococcus lactis expressing gag through oral, intranasal, subcutaneous administration or the mix of them. The mice that were immunized orally with Lactococcus lactis containing PMG36e served as a control group. The sera of mice were collected before primary immunization and 2 weeks after each immunization to detect the gag specific IgG by ELISA. Results Compared with the control group, the higher titer of serum gag specific IgG was detected in the four groups immunized with recombinant Lactococcus lactis expressing gag, and it was the highest in the mixed immunization group (P<0.01). The titer of serum gag specific IgG from the oral and subcutaneous immunization groups was significantly higher than that from the intranasal immunization group (P<0.01) 6 weeks after primary immunization . The serum antibody positive rates of the oral immunization group after the first, the second, the third immunization were 40%, 40%, 90%, respectively; the positive rates of the intranasal immunization group were 10%, 20%, 20%; the positive rates of the subcutaneous immunization group were 10%, 60%, 90%; the positive rate of the combined immunization group reached 100% 2 weeks after primary immunization. Conclusion Recombinant Lactococcus lactis expressing gag can induce humoral immune response in mice by oral, intranasal, subcutaneous injection or the mix of them, and the mixed immunization can enhance the immune effects of Lactococcus lactis vector vaccine. PMID:25374076

Zhao, Xiaofei; Zhang, Cairong; Liu, Xiaojuan; Ma, Zhenghai

2014-11-01

154

Bioelectrochemical Mn(II) leaching from manganese ore by Lactococcus lactis SK071115.  

PubMed

L. lactis sk071115 has been shown to grow more actively and generate lower levels of lactate in glucose-defined medium with nitrate than in medium with Mn(IV). By adding Mn(IV) to a L. lactis culture, lactate production was relatively reduced in combination with Mn(II) production, but cell mass production levels did not increase. Both cell-free extract and intact L. lactis cells reacted electrochemically with Mn(IV) but did not react with Mn(II) upon cyclic voltammetry using neutral red (NR) as an electron mediator. A modified graphite felt cathode with NR (NR-cathode) was employed to induce electrochemical reducing equivalence for bacterial metabolism. Cell-free L. lactis extract catalyzed the reduction of Mn(IV) to Mn(II) under both control and electrochemical reduction conditions; however, the levels of Mn(II) generated under electrochemical reduction conditions were approximately 4 times those generated under control conditions. The levels of Mn(II) generated by the catalysis of L. lactis immobilized in the NR-cathode (L-NR-cathode) under electrochemical reduction conditions were more than 4 times that generated under control conditions. Mn(II) production levels were increased by approximately 2.5 and 4.5 times by the addition of citrate to the reactant under control and electrochemical reduction conditions, respectively. The cumulative Mn(II) produced from manganese ore by catalysis of the L-NR-cathode for 30 days reached levels of approximately 3,800 and 16,000 mg/l under control and electrochemical reduction conditions, respectively. In conclusion, the electrochemical reduction reaction generated by the NR-cathode activated the biochemical reduction of Mn(IV) to Mn(II) by L. lactis. PMID:21364297

Jeon, Bo Young; Park, Doo Hyun

2011-02-01

155

Yeast on the milky way: genetics, physiology and biotechnology of Kluyveromyces lactis.  

PubMed

The milk yeast Kluyveromyces lactis has a life cycle similar to that of Saccharomyces cerevisiae and can be employed as a model eukaryote using classical genetics, such as the combination of desired traits, by crossing and tetrad analysis. Likewise, a growing set of vectors, marker cassettes and tags for fluorescence microscopy are available for manipulation by genetic engineering and investigating its basic cell biology. We here summarize these applications, as well as the current knowledge regarding its central metabolism, glucose and extracellular stress signalling pathways. A short overview on the biotechnological potential of K. lactis concludes this review. PMID:23576126

Rodicio, Rosaura; Heinisch, Jrgen J

2013-05-01

156

Mycotoxin production by isolates of Fusarium lactis from greenhouse sweet pepper (Capsicum annuum).  

PubMed

Internal fruit rot, caused by Fusarium lactis, is an important disease of sweet pepper (Capsicum annuum) in Canadian greenhouses. Production of the mycotoxins fumonisin B? (FB?), moniliformin (MON) and beauvericin (BEA) by F. lactis (17 isolates) and the related species F. proliferatum (three isolates) and F. verticillioides (one isolate), which are also associated with internal fruit rot, was evaluated on rice medium. All 21 isolates examined were found to produce BEA, at concentrations ranging from 13.28 to 1674.60 ppm, while 13 of 17 F. lactis isolates and two of three F. proliferatum isolates produced MON (0.23 to 181.85 ppm). Only one isolate of F. lactis produced detectable levels of FB? in culture, whereas all three F. proliferatum isolates and the F. verticilloides isolate produced this mycotoxin (0.28 to 314 ppm). Production of FB?, MON and BEA was also evaluated in inoculated pepper fruits showing mild or severe symptoms of infection. FB? could be detected in both lightly and heavily diseased fruit tissue after inoculation with F. lactis, F. proliferatum or F. verticilloides, at concentrations ranging from 0.61 to 8.04 ppm. BEA was also detected in lightly and heavily diseased fruit tissue inoculated with F. lactis, as well as in heavily diseased tissue inoculated with F. proliferatum (3.00 to 19.43 ppm), but not in tissue inoculated with F. verticilloides. MON was detected in all tissues inoculated with F. proliferatum or F. verticilloides, and in heavily diseased tissue inoculated with F. lactis (0.03 to 0.27 ppm). The three mycotoxins were also found in naturally infected sweet pepper fruits exhibiting symptoms of internal fruit rot and collected from a commercial greenhouse. The production of MON, BEA and FB? alone or in combination by isolates of F. lactis suggests that development of internal fruit rot of sweet pepper is an important food safety concern, and that every effort should be made to cull infected fruit before it makes it to market. PMID:21903288

Yang, Y; Bouras, N; Yang, J; Howard, R J; Strelkov, S E

2011-12-01

157

Structure and properties of the metastable bacteriocin Lcn972 from Lactococcus lactis  

NASA Astrophysics Data System (ADS)

Lactococcus lactis subsp. lactis IPLA 972 produces a polypeptide bacteriocin of 7.5 kDa which has a bactericidal effect on sensitive lactococci, inhibiting septum formation in dividing cells. The active form is a monomer that is metastable under normal conditions but is stabilised by glycerol. The NMR structure of Lcn972 shows a ?-sandwich comprising two three-stranded antiparallel ?-sheets. Detaching the final strand could allow the sandwich to open, and the irreversible unfolding leads to a loss of antibacterial activity. Covalent linkage of the final strand should increase the stability of Lcn972 and facilitate the study of its interaction with lipid II.

Turner, David L.; Lamosa, Pedro; Rodrguez, Ana; Martnez, Beatriz

2013-01-01

158

Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling  

PubMed Central

Background Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. Methodology/Principal findings We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional TH1 CD4+ and CD8+ T cells and a systemic LACK-specific TH1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific TH1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective TH1 response. Conclusions/Significance This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania. PMID:22348031

Hugentobler, Felix; Yam, Karen K.; Gillard, Joshua; Mahbuba, Raya; Olivier, Martin; Cousineau, Benoit

2012-01-01

159

A sulfur- and tyramine-regulated Klebsiella aerogenes operon containing the arylsulfatase (atsA) gene and the atsB gene.  

PubMed Central

The structural gene for arylsulfatase (atsA) of Klebsiella aerogenes was cloned into a pKI212 vector in Escherichia coli. Deletion analysis showed that the atsA gene with the promoter region was located within a 3.2-kilobase cloned segment. In E. coli cells which carried the plasmid, the synthesis of arylsulfatase was repressed by various sources of sulfur; the repression was relieved, in each case, by tyramine. Transfer of the plasmid into atsA or constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsA but not of atsR. The nucleotide sequence of the 3.2-kilobase fragment was determined. Two open reading frames, the atsA gene and an unknown gene (atsB), were found. These are located between a potential promoter and a transcriptional terminator sequence. Deletion analysis suggests that atsB is a potential positive factor for the regulation of arylsulfatase. Analysis of the amino acid sequences of the first 13 amino acids from the N terminus of the purified secreted arysulfatase agrees with that of the nucleotide sequence of atsA. The leader peptide extends over 20 amino acids and has the characteristics of a signal sequence. Primer extension mapping of transcripts generated in vivo suggests that the synthesis of mRNA starts at a site 31 or 32 bases upstream from the ATG initiation codon of the atsB gene. By Northern (RNA) blot analysis of the transcripts induced by tyramine, we found a 2.7-kilobase transcript which is identical in size to the total sequence of the atsB and atsA genes. Thus, the ats operon is composed of two cistrons and is regulated by sulfur and tyramine. Images FIG. 5 FIG. 6 PMID:2180918

Murooka, Y; Ishibashi, K; Yasumoto, M; Sasaki, M; Sugino, H; Azakami, H; Yamashita, M

1990-01-01

160

Characterization of KPC-2-producing Escherichia coli, Citrobacter freundii, Enterobacter cloacae, Enterobacter aerogenes, and Klebsiella oxytoca isolates from a Chinese Hospital.  

PubMed

Twelve nonduplicated KPC-2-producing enterobacterial isolates, including three Escherichia coli, two Citrobacter freundii, two Enterobacter cloacae, four Enterobacter aerogenes, and one Klebsiella oxytoca, were collected from various clinical samples within 18 months (March 2011 to September 2012). Two of the 12 patients died from infections caused by KPC-2-producing pathogens, while the rest of the patients with KPC-2-producing pathogens improved or were cured. The majority of the clinical isolates exhibited a high-level of resistance to oxyimino-cephalosporins and carbapenems, and possessed self-transferable bla(KPC-2)-carrying plasmids with sizes ranging from 20 to 120?kb. Most isolates carried bla(CTX-M) and plasmid-mediated quinolone resistance genes, while some isolates produced 16S rRNA methylases (ArmA or RmtB). The genetic environment of bla(KPC-2) of most clinical strains was consistent with the genetic structure surrounding bla(KPC-2) on the plasmid pKP048, which contains an integration structure of a Tn3-based transposon and partial Tn4401 segment. Inserted fragments (truncated bla(TEM)) were detected upstream of the bla(KPC-2) gene for two E. aerogenes strains. In conclusion, the enterobacterial isolates exhibited sporadic emergence and did not arise by clonal spread at our hospital. The outcome of infections caused by KPC-producing enterobacterial isolates and their mortality were closely associated with the baseline condition of patients. The spread of bla(KPC-2) gene between different enterobacterial species in China was mainly mediated by horizontal transfer of the Tn3-based transposons and not the bla(KPC-2)-carrying plasmids. PMID:24433026

Luo, Yanping; Yang, Jiyong; Ye, Liyan; Guo, Lin; Zhao, Qiang; Chen, Rong; Chen, Yong; Han, Xuelin; Zhao, Jingya; Tian, Shuguang; Han, Li

2014-08-01

161

The Amino-Terminal 100 Residues of the Nitrogen Assimilation Control Protein (NAC) Encode All Known Properties of NAC from Klebsiella aerogenes and Escherichia coli  

PubMed Central

The nitrogen assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within the nac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activate hut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACK fragment of 129 amino acids and a NACE fragment of 100 amino acids) released from comparable chimeras. A set of NACE and NACK fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NACE were insoluble, as was NACE itself. Of the his6-tagged constructs derived from NACK, NACK100 was inactive, but NACK120 was active. Several NAC fragments were tested for dimerization. NACK120-his6 and NACK100-his6 were dimers in solution. MBP-NACK and MBP-NACK129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NACE was readily cleaved by factor Xa, but the resulting NACE was also degraded by the protease. However, MBP-NACE-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein. PMID:9922258

Muse, Wilson B.; Bender, Robert A.

1999-01-01

162

Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161.  

PubMed

This work aimed at characterizing the biochemical and biophysical properties of the membrane of Lactococcus lactis TOMSC161 cells during fermentation at different temperatures, in relation to their freeze-drying and storage resistance. Cells were cultivated at two different temperatures (22 and 30C) and were harvested at different growth phases (from the middle exponential phase to the late stationary phase). Bacterial membranes were characterized by determining the fatty acid composition, the lipid phase transition, and the membrane fluidity. Cultivability and acidification activity losses of L. lactis were quantified after freezing, drying, and 3months of storage. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to lipid composition, and it was established that the cyclopropanation of unsaturated fatty acids with concomitant membrane rigidification during growth led to an increase in the freeze-drying and storage resistance of L. lactis. As expected, cultivating cells at a lower fermentation temperature than the optimum growth temperature induced a homeoviscous adaptation that was demonstrated by a lowered lipid phase transition temperature but that was not related to any improvement in freeze-drying resistance. L. lactis TOMSC161 was therefore able to develop a combined biochemical and biophysical response at the membrane level during fermentation. The ratio of cyclic fatty acids to unsaturated fatty acids (CFA/UFA) appeared to be the most relevant parameter associated with membrane rigidification and cell resistance to freeze-drying and storage. This study increased our knowledge about the physiological mechanisms that explain the resistance of lactic acid bacteria (LAB) to freeze-drying and storage stresses and demonstrated the relevance of complementary methods of membrane characterization. PMID:25343977

Velly, H; Bouix, M; Passot, S; Penicaud, C; Beinsteiner, H; Ghorbal, S; Lieben, P; Fonseca, F

2015-01-01

163

Bifidobacterium animalis ssp. lactis 420 Protects against Indomethacin-Induced Gastric Permeability in Rats  

PubMed Central

Gastrointestinal (GI) adverse effects such as erosion and increased permeability are common during the use of nonsteroidal anti-inflammatory drugs (NSAIDs). Our objective was to assess whether Bifidobacterium animalis ssp. lactis 420 protects against NSAID-induced GI side effects in a rat model. A total of 120 male Wistar rats were allocated into groups designated as control, NSAID, and probiotic. The NSAID and probiotic groups were challenged with indomethacin (10?mg/kg?1; single dose). The probiotic group was also supplemented daily with 1010?CFU of B. lactis 420 for seven days prior to the indomethacin administration. The control group rats received no indomethacin or probiotic. The permeability of the rat intestine was analysed using carbohydrate probes and the visual damage of the rat stomach mucosa was graded according to severity. B. lactis 420 significantly reduced the indomethacin-induced increase in stomach permeability. However, the protective effect on the visual mucosal damage was not significant. The incidence of severe NSAID-induced lesions was, nevertheless, reduced from 50% to 33% with the probiotic treatment. To conclude, the B. lactis 420 supplementation protected the rats from an NSAID-induced increase in stomach permeability and may reduce the formation of more serious GI mucosal damage and/or enhance the recovery rate of the stomach mucosa. PMID:22848210

Lyra, Anna; Saarinen, Markku; Putaala, Heli; Olli, Kaisa; Lahtinen, Sampo J.; Ouwehand, Arthur C.; Madetoja, Mari; Tiihonen, Kirsti

2012-01-01

164

The cmbT gene encodes a novel major facilitator multidrug resistance transporter in Lactococcus lactis.  

PubMed

Functional characterization of the multidrug resistance CmbT transporter was performed in Lactococcus lactis. The cmbT gene is predicted to encode an efflux protein homologous to the multidrug resistance major facilitator superfamily. The cmbT gene (1377 bp) was cloned and overexpressed in L. lactis NZ9000. Results from cell growth studies revealed that the CmbT protein has an effect on host cell resistance to lincomycin, cholate, sulbactam, ethidium bromide, Hoechst 33342, sulfadiazine, streptomycin, rifampicin, puromycin and sulfametoxazole. Moreover, in vivo transport assays showed that overexpressed CmbT-mediated extrusion of ethidium bromide and Hoechst 33342 was higher than in the control L. lactis NZ9000 strain. CmbT-mediated extrusion of Hoechst 33342 was inhibited by the ionophores nigericin and valinomycin known to dissipate proton motive force. This indicates that CmbT-mediated extrusion is based on a drug-proton antiport mechanism. Taking together results obtained in this study, it can be concluded that CmbT is a novel major facilitator multidrug resistance transporter candidate in L. lactis, with a possible signaling role in sulfur metabolism. PMID:22985829

Filipic, Brankica; Golic, Natasa; Jovcic, Branko; Tolinacki, Maja; Bay, Denice C; Turner, Raymond J; Antic-Stankovic, Jelena; Kojic, Milan; Topisirovic, Ljubisa

2013-01-01

165

Comparative Analyses of Prophage-Like Elements Present in Two Lactococcus lactis Strains?  

PubMed Central

In this study, we describe the genetic organizations of six and five apparent prophage-like elements present in the genomes of the Lactococcus lactis subsp. cremoris strains MG1363 and SK11, respectively. Phylogenetic investigation as well bioinformatic analyses indicates that all 11 prophages belong to subdivisions of the lactococcal P335 group of temperate bacteriophages. PMID:17933937

Ventura, Marco; Zomer, Aldert; Canchaya, Carlos; O'Connell-Motherway, Mary; Kuipers, Oscar; Turroni, Francesca; Ribbera, Angela; Foroni, Elena; Buist, Girbe; Wegmann, Udo; Shearman, Claire; Gasson, Michael J.; Fitzgerald, Gerald F.; Kok, Jan; van Sinderen, Douwe

2007-01-01

166

Sulfur Amino Acid Metabolism and Its Control in Lactococcus lactis IL1403  

Microsoft Academic Search

Cysteine and methionine availability influences many processes in the cell. In bacteria, transcription of the specific genes involved in the synthesis of these two amino acids is usually regulated by different mechanisms or regulators. Pathways for the synthesis of cysteine and methionine and their interconversion were experi- mentally determined for Lactococcus lactis, a lactic acid bacterium commonly found in food.

Brice Sperandio; Patrice Polard; Dusko S. Ehrlich; Pierre Renault; Eric Guedon

2005-01-01

167

Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications  

Microsoft Academic Search

BACKGROUND: The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) produced by

Igor Mierau; Kees Olieman; James Mond; Eddy J Smid

2005-01-01

168

Development, molecular characterization and exploitation of the nisin controlled expression system in Lactococcus lactis  

Microsoft Academic Search

Lactic acid bacteria are gram-positive bacteria that are widely used in a variety of dairy fermentation processes. Notably, strains of the lactic acid starter bacterium Lactococcus lactis are of great economic importance because of their world-wide use in cheese making. The characteristic aroma, flavor and texture of cheese develops during ripening of the cheese curd through the action of numerous

Ruyter de P. G. G. A

1998-01-01

169

Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions  

PubMed Central

Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. lactis subsp. cremoris MG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L. lactis, mimicking as closely as practically possible its intracellular environment. Procedures were optimized to be carried out in 96-well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established. Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of Vmax over measured in vivo fluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements for L. lactis. PMID:22020503

Goel, Anisha; Santos, Filipe; de Vos, Willem M.; Teusink, Bas

2012-01-01

170

Complete Genome Sequence of Lactococcus lactis Strain AI06, an Endophyte of the Amazonian Aa Palm.  

PubMed

We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the aa fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the aa palm and also a component of the microbiota of the edible food product. PMID:25414513

McCulloch, John Anthony; de Oliveira, Viviane Matoso; de Almeida Pina, Andr Vicioli; Prez-Chaparro, Paula Juliana; de Almeida, Lara Mendes; de Vasconcelos, Janaina Mota; de Oliveira, Layanna Freitas; da Silva, Daisy Elaine Andrade; Rogez, Herv Louis Ghislain; Cretenet, Marina; Mamizuka, Elsa Masae; Nunes, Marcio Roberto Teixeira

2014-01-01

171

Complete Genome Sequence of Lactococcus lactis Strain AI06, an Endophyte of the Amazonian Aa Palm  

PubMed Central

We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the aa fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the aa palm and also a component of the microbiota of the edible food product. PMID:25414513

de Oliveira, Viviane Matoso; de Almeida Pina, Andr Vicioli; Prez-Chaparro, Paula Juliana; de Almeida, Lara Mendes; de Vasconcelos, Janaina Mota; de Oliveira, Layanna Freitas; da Silva, Daisy Elaine Andrade; Rogez, Herv Louis Ghislain; Cretenet, Marina; Mamizuka, Elsa Masae; Nunes, Marcio Roberto Teixeira

2014-01-01

172

Kinetics and regulation of lactose transport and metabolism in Kluyveromyces lactis JA6.  

PubMed

Kluyveromyces lactis strains are able to assimilate lactose. They have been used industrially to eliminate this sugar from cheese whey and in other industrial products. In this study, we investigated specific features and the kinetic parameters of the lactose transport system in K. lactis JA6. In lactose grown cells, lactose was transported by a system transport with a half-saturation constant (K s) of 1.49 0.38 mM and a maximum velocity (V max) of 0.96 0.12 mmol. (g dry weight)(-1) h(-1) for lactose. The transport system was constitutive and energy-dependent. Results obtained by different approaches showed that the lactose transport system was regulated by glucose at the transcriptional level and by glucose and other sugars at a post-translational level. In K. lactis JA6, galactose metabolization was under glucose control. These findings indicated that the regulation of lactose-galactose regulon in K. lactis was similar to the regulation of galactose regulon in Saccharomyces cerevisiae. PMID:24504708

Santos, A M; Silveira, W B; Fietto, L G; Brando, R L; Castro, I M

2014-07-01

173

The Plasmid Complement of Lactococcus lactis UC509.9 Encodes Multiple Bacteriophage Resistance Systems  

PubMed Central

Lactococcus lactis subsp. cremoris strains are used globally for the production of fermented dairy products, particularly hard cheeses. Believed to be of plant origin, L. lactis strains that are used as starter cultures have undergone extensive adaptation to the dairy environment, partially through the acquisition of extrachromosomal DNA in the form of plasmids that specify technologically important phenotypic traits. Here, we present a detailed analysis of the eight plasmids of L. lactis UC509.9, an Irish dairy starter strain. Key industrial phenotypes were mapped, and genes that are typically associated with lactococcal plasmids were identified. Four distinct, plasmid-borne bacteriophage resistance systems were identified, including two abortive infection systems, AbiB and AbiD1, thereby supporting the observed phage resistance of L. lactis UC509.9. AbiB escape mutants were generated for phage sk1, which were found to carry mutations in orf6, which encodes the major capsid protein of this phage. PMID:24814781

Ainsworth, Stuart; Mahony, Jennifer

2014-01-01

174

Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures  

PubMed Central

A large collection of Lactococcus lactis strains, including wild-type isolates and dairy starter cultures, were screened on the basis of their phenotype and the macrorestriction patterns produced from pulsed-field gel electrophoresis (PFGE) analysis of SmaI digests of genomic DNA. Three groups of dairy starter cultures, used for different purposes in the dairy industry, and a fourth group made up of strains isolated from the environment were selected for analysis of their chromosomal diversity using the endonuclease I-CeuI. Chromosome architecture was largely conserved with each strain having six copies of the rRNA genes, and the chromosome size of individual strains ranged between 2,240 and 2,688 kb. The origin of L. lactis strains showed the greatest correlation with chromosome size, and dairy strains, particularly those with the cremoris phenotype, had smaller chromosomes than wild-type strains. Overall, this study, coupled with analysis of the sequenced L. lactis genomes, provides evidence that defined strain dairy starter cultures have arisen from plant L. lactis strains. Adaptation of these strains to the dairy environment has involved loss of functions resulting in smaller chromosomes and acquisition of genes (usually plasmid associated) that facilitate growth in milk. We conclude that dairy starter cultures generally and the industrially used cremoris and diacetylactis phenotype strains in particular comprise a specialized group of L. lactis strains that have been selected to become an essential component of industrial processes and have evolved accordingly, so that they are no longer fit to survive outside the dairy environment. PMID:20847124

Kelly, William J.; Ward, Lawrence J. H.; Leahy, Sinead C.

2010-01-01

175

Lactococcus lactis subsp. cremoris FC alleviates symptoms of colitis induced by dextran sulfate sodium in mice.  

PubMed

Probiotics have been used to treat human gastrointestinal inflammations including inflammatory bowel disease (IBD). However, the exact mechanisms by which probiotics act to protect against intestinal inflammation have yet to be fully elucidated. The aim of this study was to evaluate anti-inflammatory effects of Lactococcus lactis subsp. cremoris FC using in vivo and in vitro inflammation models. Colitis was induced in C57BL/6 mice by administration of 3% dextran sulfate sodium to drinking water. In the cellular level assessment, a gut inflammation model with the co-culture system consisting Caco-2 cells and RAW264.7 cells stimulated by LPS was used. Administration of L. lactis subsp. cremoris FC significantly ameliorated shortening of colon length and histological score of the colon in DSS-induce colitis mice. In addition, the treatment of L. lactis subsp. cremoris FC improved the aberrant mRNA expression in inflamed tissue near to control level through notable suppression of TNF-alpha (P<0.05), IFN-gamma (P<0.05), IL-6, iNOS, and MIP-2 mRNA expression. In addition, in a gut inflammation model, treatment with L. lactis subsp. cremoris FC resulted in significant down-regulation of IL-8 mRNA expression in Caco-2 cells and inhibition of NF-kappaB nuclear translocation in RAW264.7 cells. Our findings indicate that administration of L. lactis subsp. cremoris FC improves negative effects of DSS-induced colitis in mice through the inhibition of inflammatory cell infiltration. PMID:19733697

Nishitani, Yosuke; Tanoue, Takeshi; Yamada, Katsushige; Ishida, Tsukasa; Yoshida, Masaru; Azuma, Takeshi; Mizuno, Masashi

2009-11-01

176

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour  

Microsoft Academic Search

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well\\u000a as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions\\u000a were used to fit and verify

C. kerberg; K. Hofvendahl; G. Zacchi; B. Hahn-Hgerdal

1998-01-01

177

Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis.  

PubMed

Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications. PMID:25447786

Sheng, Juzheng; Ling, Peixue; Wang, Fengshan

2014-12-01

178

Mdt(A), a New Efflux Protein Conferring Multiple Antibiotic Resistance in Lactococcus lactis and Escherichia coli  

Microsoft Academic Search

The mdt(A) gene, previously designated mef214, from Lactococcus lactis subsp. lactis plasmid pK214 encodes a protein (Mdt(A) (multiple drug transporter)) with 12 putative transmembrane segments (TMS) that contain typical motifs conserved among the efflux proteins of the major facilitator superfamily. However, it also has two C-motifs (conserved in the fifth TMS of the antiporters) and a putative ATP-binding site. Expression

VINCENT PERRETEN; FRANZISKA V. SCHWARZ; MICHAEL TEUBER; STUART B. LEVY

2001-01-01

179

Effects of strains of Lactococcus lactis on the production of nitric oxide and cytokines in murine macrophages.  

PubMed

Nitric oxide (NO) is a multifunctional mediator that is involved in a variety of pathologic and physiologic processes. Few studies have addressed the effect of lactic acid bacteria (LAB), especially Lactococcus lactis strains used in dairy products, on inducible nitric oxide synthase (iNOS) induction as a component of the host's gastrointestinal immune response. We investigated the ability of L. lactis strains to induce NO synthesis in the murine macrophage-like cell line J774.1 and in peritoneal macrophages from mice. The degree of NO induction was specific to the L. lactis strain used. Compared with the no-treatment control, heat treatment of L. lactis cells decreased NO and TNF-? levels but further stimulated interleukin (IL)-12 production. Adding L. lactis cells to peritoneal macrophages dose-dependently increased the production of NO and IL-10 but decreased that of IL-12p70. Adding L. lactis cells to interferon-?-stimulated J774.1 cells enhanced cell death and the production of NO and IL-12p40, whereas addition of 1400W, a specific inhibitor of iNOS, decreased NO production and cell death. Conversely, adding 1400W to J774.1 cells further enhanced IL-12p40 production, suggesting that IL-12 production is perturbed by excess endogenous NO. IL-12 production is thought to be a marker of improved immunostimulation. Our results suggest that IL-12 production could be increased by limiting endogenous NO production. PMID:24818707

Suzuki, Chise; Aoki-Yoshida, Ayako; Kimoto-Nira, Hiromi; Kobayashi, Miho; Sasaki, Keisuke; Mizumachi, Koko

2014-10-01

180

Metabolic engineering of Kluyveromyces lactis for L-ascorbic acid (vitamin C) biosynthesis  

PubMed Central

Background L-ascorbic acid (L-AA) is naturally synthesized in plants from D-glucose by 10 steps pathway. The pathway branch to synthesize L-galactose, the key intermediate for L-ascorbic acid biosynthesis, has been recently elucidated. Budding yeast produces an 5-carbon ascorbic acid analogue Dehydro-D-arabinono 1,4-lactone (D-DAL), which is synthesized from D-arabinose. Yeast is able to synthesize L-ascorbic acid only if it is cultivated in the presence of one of its precursors: L-galactose, L-galactono 1,4-lactone, or L-gulono 1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this L enantiomer, we engineered Kluyveromyces lactis with L-galactose biosynthesis pathway genes: GDP-mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1-phosphate phosphatase (VTC4) isolated from Arabidopsis thaliana. Results Plasmids were constructed and modified such that the cloned plant genes were targeted to the K. lactis LAC4 Locus by homologous recombination and that the expression was associated to the growth on D-galactose or lactose. Upon K. lactis transformation, GME was under the control of the native LAC4 promoter whereas VTC2 and VTC4 were expressed from the S. cerevisiae promoters GPD1 and ADH1 respectively. The expression in K. lactis, of the L-galactose biosynthesis genes was determined by Reverse Transcriptase-PCR and western blotting. The recombinant yeasts were capable to produce about 30mg.L-1 of L-ascorbic acid in 48hours of cultivation when cultured on rich medium with 2% (w/v) D-galactose. We also evaluated the L-AA production culturing recombinant recombinant strains in cheese whey, a waste product during cheese production, as an alternative source of lactose. Conclusions This work is the first attempt to engineer K. lactis cells for L-ascorbic acid biosynthesis by a fermentation process without any trace of L isomers precursors in the culture medium. We have engineered K. lactis strains capable of converting lactose and D-galactose into L-galactose, by the integration of the genes from the A. thaliana L-galactose pathway. L-galactose is a rare sugar, which is one of the main precursors for L-AA production. PMID:23799937

2013-01-01

181

1 JUNE 2006 Bact to basics  

E-print Network

, forming an electric current. Courtesy Geobacter Project That's only slightly below the level industrial fermenters get while using bacteria to make ethanol for car fuel. But B subtilis has some advantages's called getting a two-fer. Bacteria battery Instead of using bacteria to make fuel, what about using them

Lovley, Derek

182

Characterization of the second external alternative dehydrogenase from mitochondria of the respiratory yeast Kluyveromyces lactis.  

PubMed

The mitochondria of the respiratory yeast Kluyveromyces lactis are able to reoxidize cytosolic NADPH. Previously, we characterized an external alternative dehydrogenase, KlNde1p, having this activity. We now characterize the second external alternative dehydrogenase of K. lactis mitochondria, KlNde2p. We examined its role in cytosolic NADPH reoxidation by studying heterologous expression of KlNDE2 in Saccharomyces cerevisiae mutants and by constructing Deltaklnde1 and Deltaklnde2 mutants. KlNde2p uses NADH or NADPH as substrates, its activity in isolated mitochondria is not regulated by exogenously added calcium and it is not down-regulated when the cells grow in glucose versus lactate. KlNde2p shows lower affinity for NADPH than KlNde1p. Both enzymes show similar pH optimum. PMID:17052684

Tarro, Nuria; Cerdn, M Esperanza; Gonzlez Siso, M Isabel

2006-11-01

183

Defined bacterial culture development for methane generation from lactose. [Streptococcus lactis; Clostridium formicoaceticum; Methanococcus mazei  

SciTech Connect

The defined microbial cultures for methane generation from lactose were investigated. A mixed culture consisting of homolactic (Streptococcus lactis), homoacetic (Clostridium formicoaceticum), and acetate-utilizing methanogenic (Methanococcus mazei) bacteria was used to convert lactose and whey permeate to methane at mesophilic temperatures (35-37/sup 0/C) and a pH around 7.0. Lactose was first converted to lactic acid by S. lactis, then to acetic acid by C. formicoaceticum, and finally to methane and CO/sub 2/ by M. mazei. About 5.3 mol methane were obtained from each mole of lactose consumed, and the conversion of acetate to methane was the rate-limiting step for this mixed-culture fermentation.

Yang, S.T.; Tang, I.C.; Okos, M.R.

1988-06-20

184

A dynamic, genome-scale flux model of Lactococcus lactis to increase specific recombinant protein expression.  

PubMed

Recently, lactic acid bacteria (LAB) have attracted a great deal of interest because of their potential to serve as oral delivery vehicles for recombinant protein vaccines. An important limitation to their use is the typically low level of heterologous expression obtained in LAB. To address this, a dynamic flux balance analysis (DFBA) model was used to identify gene targets for increasing specific expression of Green Fluorescent Protein (GFP), a model heterologous protein, in Lactococcus lactis IL1403. Two strains, each targeting one of the top model-identified genes, were constructed and tested in vivo. Data show that both strains, by a conservative estimate, achieved 15% higher GFP per cell than the control strain, a qualitative confirmation of the model predictions. A genome-scale DFBA model for L. lactis growing on M17 medium is presented along with the procedure for screening gene targets and a powerful method for visualizing fluxes in genome-scale metabolic networks. PMID:19666133

Oddone, Gian M; Mills, David A; Block, David E

2009-11-01

185

Construction and application of a food-grade expression system for Lactococcus lactis.  

PubMed

A food-grade host/vector expression system for Lactococcus lactis was constructed using alanine racemase gene (alr) as the complementation marker. We obtained an alanine racemase auxotrophic mutant L. lactis NZ9000?alr by double-crossover recombination using temperature-sensitive integration plasmid pG(+)host9 and a food-grade vector pALR with entirely lactococcal DNA elements, including lactococcal replicon, nisin-inducible promoter PnisA and the alr gene from Lactobacillus casei BL23 as a complementation marker. By using the new food-grade host/vector system, the green fluorescent protein and capsid protein of porcine circovirus type II were successfully overexpressed under the nisin induction. These results indicate that this food-grade host/vector expression system has application potential as an excellent antigen delivery vehicle, and is also suitable for the use in the manufacture of ingredients for the food industry. PMID:22674186

Lu, Wenwei; Kong, Jian; Kong, Wentao

2013-06-01

186

Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis  

PubMed Central

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters. PMID:23144255

Trip, Hein; Mulder, Niels L.

2013-01-01

187

Food-grade controlled lysis of Lactococcus lactis for accelerated cheese ripening  

Microsoft Academic Search

An attractive approach to accelerate cheese ripening is to induce lysis of Lactococcus lactis starter strains for facilitated release of intracellular enzymes involved in flavor formation. Controlled expression of the lytic genes lytA and lytH, which encode the lysin and the holin proteins of the lactococcal bacteriophage ?US3, respectively, was accomplished by application of a food-grade nisin-inducible expression system. Simultaneous

Oscar P. Kuipers; Wilco C. Meijer; Willem M. de Vos

1997-01-01

188

Feedback Regulation of Glucose Transporter Gene Transcription in Kluyveromyces lactis by Glucose Uptake  

Microsoft Academic Search

In the respirofermentative yeast Kluyveromyces lactis, only a single genetic locus encodes glucose transporters that can support fermentative growth. This locus is polymorphic in wild-type isolates carrying either KHT1 and KHT2, two tandemly arranged HXT-like genes, or RAG1, a low-affinity transporter gene that arose by recom- bination between KHT1 and KHT2. Here we show that KHT1 is a glucose-induced gene

C. Milkowski; S. Krampe; J. Weirich; V. Hasse; E. Boles; K. D. Breunig

2001-01-01

189

?-Acetolactate synthase of Lactococcus lactis contributes to pH homeostasis in acid stress conditions.  

PubMed

Lactic Acid Bacteria (LAB) are recognized as safe microorganisms with the capacity to improve the quality of dairy products. When the LAB Lactococcus lactis is employed as starter for the production of fermented foods, high quantities of important aroma compounds such as diacetyl are generated by means of the diacetyl/acetoin pathway. Our previous results obtained with L. lactis strains report that this pathway is activated under acidic conditions. In this study, we describe the metabolism of pyruvate, a diacetyl/acetoin precursor, and its contribution to pH homeostasis in this microorganism. L lactis strain IL1403 is able to cometabolize pyruvate and glucose at low pH, producing lactate, acetate as well as diacetyl/acetoin compounds. In contrast, the als defective strain, which is incapable of producing C4 compounds, appeared sensitive to pyruvate under acidic conditions rendering it unable to grow. Accordingly, the als-mutant strain showed a simultaneous inability to alkalinize internal and external media. These results demonstrate that the decarboxylation reactions associated to the diacetyl/acetoin pathway represent a competitive advantage in a condition of intracellular pyruvate accumulation during growth at low pH. Interestingly, a genomic comparative analysis shows that this pathway has been conserved in L. lactis during the domestication of different strains. Also, our analysis shows that the recent acquisition of the cit cluster required for citrate metabolism, which contributes to diacetyl/acetoin production as well, is the specific feature of the biovar. diacetylactis. In this regard, we present for first time genetic evidence supporting the proposal made by Passerini et al. (2013) who postulated that the expression "biovar. citrate" should be more appropriate to define this specific industrial strain. PMID:25100661

Zuljan, Federico A; Repizo, Guillermo D; Alarcon, Sergio H; Magni, Christian

2014-10-01

190

Production of a Particulate Hepatitis C Vaccine Candidate by an Engineered Lactococcus lactis Strain?  

PubMed Central

Vaccine delivery systems based on display of antigens on bioengineered bacterial polyester inclusions can stimulate cellular immune responses. The food-grade Gram-positive bacterium Lactococcus lactis was engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which abundantly displayed the hepatitis C virus core (HCc) antigen. In mice, the immune response induced by this antigen delivery system was compared to that induced by vaccination with HCc antigen displayed on PHB beads produced in Escherichia coli, to PHB beads without antigen produced in L. lactis or E. coli, or directly to the recombinant HCc protein. Vaccination site lesions were minimal in all mice vaccinated with HCc PHB beads or recombinant protein, all mixed in the oil-in-water adjuvant Emulsigen, while vaccination with the recombinant protein in complete Freund's adjuvant produced a marked inflammatory reaction at the vaccination site. Vaccination with the PHB beads produced in L. lactis and displaying HCc antigen produced antigen-specific cellular immune responses with significant release of gamma interferon (IFN-?) and interleukin-17A (IL-17A) from splenocyte cultures and no significant antigen-specific serum antibody, while the PHB beads displaying HCc but produced in E. coli released IFN-? and IL-17A as well as the proinflammatory cytokines tumor necrosis factor alpha (TNF-?) and IL-6 and low levels of IgG2c antibody. In contrast, recombinant HCc antigen in Emulsigen produced a diverse cytokine response and a strong IgG1 antibody response. Overall it was shown that L. lactis can be used to produce immunogenic PHB beads displaying viral antigens, making the beads suitable for vaccination against viral infections. PMID:21984246

Parlane, Natalie A.; Grage, Katrin; Lee, Jason W.; Buddle, Bryce M.; Denis, Michel; Rehm, Bernd H. A.

2011-01-01

191

Characterization of the Lactococcus lactis lactose genes and regulation of their expression  

Microsoft Academic Search

An important trait of the lactic acid bacterium Lactococcus lactis , that is used in industrial dairy fermentations, is the conversion of lactose into lactic acid. The enzymatic steps involved in the breakdown of lactose, that is transported into the cell via a phosphoenolpyruvate-dependent lactose phosphotransferase system (PEP-PTS lac<\\/SUP>), have been well established (Fig. 1). However, except for the molecular

Rooijen van R. J

1993-01-01

192

Glycolic acid production in the engineered yeasts Saccharomyces cerevisiae and Kluyveromyces lactis  

PubMed Central

Background Glycolic acid is a C2 hydroxy acid that is a widely used chemical compound. It can be polymerised to produce biodegradable polymers with excellent gas barrier properties. Currently, glycolic acid is produced in a chemical process using fossil resources and toxic chemicals. Biotechnological production of glycolic acid using renewable resources is a desirable alternative. Results The yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are suitable organisms for glycolic acid production since they are acid tolerant and can grow in the presence of up to 50gl-1 glycolic acid. We engineered S. cerevisiae and K. lactis for glycolic acid production using the reactions of the glyoxylate cycle to produce glyoxylic acid and then reducing it to glycolic acid. The expression of a high affinity glyoxylate reductase alone already led to glycolic acid production. The production was further improved by deleting genes encoding malate synthase and the cytosolic form of isocitrate dehydrogenase. The engineered S. cerevisiae strain produced up to about 1gl-1 of glycolic acid in a medium containing d-xylose and ethanol. Similar modifications in K. lactis resulted in a much higher glycolic acid titer. In a bioreactor cultivation with d-xylose and ethanol up to 15gl-1 of glycolic acid was obtained. Conclusions This is the first demonstration of engineering yeast to produce glycolic acid. Prior to this work glycolic acid production through the glyoxylate cycle has only been reported in bacteria. The benefit of a yeast host is the possibility for glycolic acid production also at low pH, which was demonstrated in flask cultivations. Production of glycolic acid was first shown in S. cerevisiae. To test whether a Crabtree negative yeast would be better suited for glycolic acid production we engineered K. lactis in the same way and demonstrated it to be a better host for glycolic acid production. PMID:24053654

2013-01-01

193

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.  

PubMed Central

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase. Images PMID:1539995

Bruinenberg, P G; Vos, P; De Vos, W M

1992-01-01

194

PpiA, a Surface PPIase of the Cyclophilin Family in Lactococcus lactis  

PubMed Central

Background Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. Results In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H2O2) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H2O2. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. Conclusions Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro. PMID:22442694

Trmillon, Nicolas; Morello, Eric; Llull, Daniel; Mazmouz, Rabia; Gratadoux, Jean-Jacques; Guillot, Alain; Chapot-Chartier, Marie-Pierre; Monlezun, Laura; Sol, Vronique; Ginisty, Herv; Poquet, Isabelle

2012-01-01

195

Characterization of the CopR Regulon of Lactococcus lactis IL1403  

Microsoft Academic Search

To identify components of the copper homeostatic mechanism of Lactococcus lactis, we employed two- dimensional gel electrophoresis to detect changes in the proteome in response to copper. Three proteins upregulated by copper were identified: glyoxylase I (YaiA), a nitroreductase (YtjD), and lactate oxidase (LctO). The promoter regions of these genes feature cop boxes of consensus TACAnnTGTA, which are the binding

David Magnani; Olivier Barre; Simon D. Gerber; Marc Solioz

2008-01-01

196

Enhanced nutty flavor formation in cheddar cheese made with a malty Lactococcus lactis adjunct culture.  

PubMed

Nutty flavor in Cheddar cheese is desirable, and recent research demonstrated that 2- and 3-methyl butanal and 2-methyl propanal were primary sources of nutty flavors in Cheddar. Because malty strains of Lac-tococcus lactis (formerly Streptococcus lactis var. malti-genes) are characterized by the efficient production of these and other Strecker aldehydes during growth, this study investigated the influence of a malty L. lactis adjunct culture on nutty flavor development in Cheddar cheese. Cheeses made with different adjunct levels (0, 10(4) cfu/mL, and 10(5) cfu/mL) were ripened at 5 or 13 degrees C and analyzed after 1 wk, 4 mo, and 8 mo by a combination of instrumental and sensory methods to characterize nutty flavor development. Cheeses ripened at 13 degrees C developed aged flavors (brothy, sulfur, and nutty flavors) more rapidly than cheeses held at 5 degrees C. Additionally, cheeses made with the adjunct culture showed more rapid and more intense nutty flavor development than control cheeses. Cheeses that had higher intensities of nutty flavors also had a higher concentration of 2/3-methyl butanal and 2-methyl propanal compared with control cheeses, which again confirmed that these compounds are a source of nutty flavor in Cheddar cheese. Results from this study provide a simple methodology for cheese manufacturers to obtain consistent nutty flavor in Cheddar cheese. PMID:16899660

Whetstine, M E Carunchia; Drake, M A; Broadbent, J R; McMahon, D

2006-09-01

197

Glucose metabolism and ethanol production in adh multiple and null mutants of Kluyveromyces lactis.  

PubMed

Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten-fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen-limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants. PMID:7754703

Saliola, M; Bellardi, S; Marta, I; Falcone, C

1994-09-01

198

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin  

PubMed Central

Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37C than at 30C. These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen. PMID:23913422

Chen, Jun; Shen, Jing

2013-01-01

199

Microencapsulation of Bifidobacterium animalis subsp. lactis and Lactobacillus acidophilus in cocoa butter using spray chilling technology  

PubMed Central

In the present study, the cells of Bifidobacterium animalis subsp. lactis (BI-01) and Lactobacillus acidophilus (LAC-04) were encapsulated in cocoa butter using spray-chilling technology. Survival assays were conducted to evaluate the resistance of the probiotics to the spray-chilling process, their resistance to the simulated gastric and intestinal fluids (SGF and SIF), and their stability during 90 days of storage. The viability of the cells was not affected by microencapsulation. The free and encapsulated cells of B. animalis subsp. lactis were resistant to both SGF and SIF. The micro-encapsulated cells of L. acidophilus were more resistant to SGF and SIF than the free cells; the viability of the encapsulated cells was enhanced by 67%, while the free cells reached the detection limit of the method (103 CFU/g). The encapsulated probiotics were unstable when they were stored at 20 C. The population of encapsulated L. acidophilus decreased drastically when they were stored at 7 C; only 20% of cells were viable after 90 days of storage. The percentage of viable cells of the encapsulated B. animalis subsp.lactis, however, was 72% after the same period of storage. Promising results were obtained when the microparticles were stored at ?18 C; the freeze granted 90 days of shelf life to the encapsulated cells. These results suggest that the spray-chilling process using cocoa butter as carrier protects L. acidophilus from gastrointestinal fluids. However, the viability of the cells during storage must be improved. PMID:24516445

Pedroso, D.L.; Dogenski, M.; Thomazini, M.; Heinemann, R.J.B.; Favaro-Trindade, C.S.

2013-01-01

200

Sec-mediated transport of posttranslationally dehydrated peptides in Lactococcus lactis.  

PubMed

Nisin is a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis. Its (methyl)lanthionines are introduced by two posttranslational enzymatic steps involving the dehydratase NisB, which dehydrates serine and threonine residues, and the cyclase NisC, which couples these dehydrated residues to cysteines, yielding thioether-bridged amino acids called lanthionines. The prenisin is subsequently exported by the ABC transporter NisT and extracellularly processed by the peptidase NisP. L. lactis expressing the nisBTC genes can modify and secrete a wide range of nonlantibiotic peptides. Here we demonstrate that in the absence of NisT and NisC, the Sec pathway of L. lactis can be exploited for the secretion of dehydrated variants of therapeutic peptides. Furthermore, posttranslational modifications by NisB and NisC still occur even when the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively. However, transport of fully modified prenisin via the Sec pathway is impaired. The extent of NisB-mediated dehydration could be improved by raising the intracellular concentration NisB or by modulating the export efficiency through altering the signal sequence. These data demonstrate that besides the traditional lantibiotic transporter NisT, the Sec pathway with an established broad substrate range can be utilized for the improved export of lantibiotic enzyme-modified (poly)peptides. PMID:17041158

Kuipers, Anneke; Wierenga, Jenny; Rink, Rick; Kluskens, Leon D; Driessen, Arnold J M; Kuipers, Oscar P; Moll, Gert N

2006-12-01

201

Regulation of Acetate Kinase Isozymes and Its Importance for Mixed-Acid Fermentation in Lactococcus lactis  

PubMed Central

Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The Km values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate. PMID:24464460

Puri, Pranav; Goel, Anisha; Bochynska, Agnieszka

2014-01-01

202

A monoamine-regulated Klebsiella aerogenes operon containing the monoamine oxidase structural gene (maoA) and the maoC gene.  

PubMed Central

The Klebsiella aerogenes gene maoA, which is involved in the synthesis of monoamine oxidase, was induced by tyramine and the related compounds, subjected to catabolite and ammonium ion repression, and cloned. The nucleotide sequence of the region involved in monoamine oxidase synthesis was determined. Two open reading frames, the maoA gene and a hitherto unknown gene (maoC), were found. These are located between a potential promoter sequence and a transcriptional terminator sequence. A region of the Escherichia coli chromosome that was highly homologous to the Klebsiella maoA gene was found. The potential maoA gene is located at 30.9 min on the E. coli chromosome. Analysis of the amino acid sequences of the first 11 amino acids from the N terminus of the purified monoamine oxidase agrees with those deduced from the nucleotide sequence of the maoA gene. The leader peptide extends over 30 amino acids and has the characteristics of a signal sequence. Primer extension and S1 nuclease mapping of transcripts generated in vivo suggests that the tyramine-induced mRNA starts at a site 62 bases upstream from the ATG initiation codon of the maoC gene. In the putative promoter region, a high degree of similarity to the consensus sequence for the binding site of cyclic AMP receptor protein was found. Thus, the mao region is composed of two cistrons, and the mao operon is regulated by monoamine compounds, glucose, and ammonium ions. Images PMID:1556068

Sugino, H; Sasaki, M; Azakami, H; Yamashita, M; Murooka, Y

1992-01-01

203

Malonate-bound structure of the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) and characterization of the native Fe[superscript 2+] metal-ion preference  

SciTech Connect

The structure of a malonate-bound form of the glycerophosphodiesterase from Enterobacter aerogenes, GpdQ, has been refined at a resolution of 2.2 {angstrom} to a final R factor of 17.1%. The structure was originally solved to 2.9 {angstrom} resolution using SAD phases from Zn{sup 2+} metal ions introduced into the active site of the apoenzyme [Jackson et al. (2007), J. Mol. Biol. 367, 1047-1062]. However, the 2.9 {angstrom} resolution was insufficient to discern significant details of the architecture of the binuclear metal centre that constitutes the active site. Furthermore, kinetic analysis revealed that the enzyme lost a significant amount of activity in the presence of Zn2+, suggesting that it is unlikely to be a catalytically relevant metal ion. In this communication, a higher resolution structure of GpdQ is presented in which malonate is visibly coordinated in the active site and analysis of the native metal-ion preference is presented using atomic absorption spectroscopy and anomalous scattering. Catalytic implications of the structure and its Fe{sup 2+} metal-ion preference are discussed.

Jackson, Colin J.; Hadler, Kieran S.; Carr, Paul D.; Oakley, Aaron J.; Yip, Sylvia; Schenk, Gerhard; Ollis, David L. (Queensland); (ANU)

2011-09-28

204

Characterization of In40 of Enterobacter aerogenes BM2688, a Class 1 Integron with Two New Gene Cassettes, cmlA2 and qacF  

PubMed Central

Enterobacter aerogenes BM2688, which is resistant to multiple antibiotics, and its aminoglycoside-susceptible derivative BM2688-1 were isolated from the same clinical sample. Strain BM2688 harbored plasmid pIP833, which carries a class 1 integron, In40, containing (in addition to qacE?1 and sul1, which are characteristic of class 1 integrons) four gene cassettes: aac(6?)-Ib, qacF, cmlA2, and oxa-9. The cmlA2 gene had 83.7% identity with the previously described nonenzymatic chloramphenicol resistance cmlA1 gene. The qacF gene conferred resistance to quaternary ammonium compounds and displayed a high degree of similarity with qacE (67.8% identity) which, however, has been found as part of a cassette with a very different 59-base element. The oxa-9 gene was not expressed due to a lack of promoter sequences. Study of the antibiotic-susceptible derivative BM2688-1 indicated that a 3,148-bp deletion between the 3? end of the aac(6?)-Ib gene and the 3? conserved segment of In40 was responsible for the loss of resistance. The occurrence of this DNA rearrangement, which did not involve homologous sequences, suggests that the In40 integrase could promote recombination at secondary sites. PMID:9756755

Ploy, Marie-Ccile; Courvalin, Patrice; Lambert, Thierry

1998-01-01

205

Purification and characterization of Klebsiella aerogenes UreE protein: a nickel-binding protein that functions in urease metallocenter assembly.  

PubMed Central

The Klebsiella aerogenes ureE gene product was previously shown to facilitate assembly of the urease metallocenter (Lee, M.H., et al., 1992, J. Bacteriol. 174, 4324-4330). UreE protein has now been purified and characterized. Although it behaves as a soluble protein, UreE is predicted to possess an amphipathic beta-strand and exhibits unusually tight binding to phenyl-Sepharose resin. Immunogold electron microscopic studies confirm that UreE is a cytoplasmic protein. Each dimeric UreE molecule (M(r) = 35,000) binds 6.05 + 0.25 nickel ions (Kd of 9.6 +/- 1.3 microM) with high specificity according to equilibrium dialysis measurements. The nickel site in UreE was probed by X-ray absorption and variable-temperature magnetic circular dichroism spectroscopies. The data are most consistent with the presence of Ni(II) in pseudo-octahedral geometry with 3-5 histidyl imidazole ligands. The remaining ligands are nitrogen or oxygen donors. UreE apoprotein has been crystallized and analyzed by X-ray diffraction methods. Addition of nickel ion to apoprotein crystals leads to the development of fractures, consistent with a conformational change upon binding nickel ion. We hypothesize that UreE binds intracellular nickel ion and functions as a nickel donor during metallocenter assembly into the urease apoprotein. PMID:8318889

Lee, M. H.; Pankratz, H. S.; Wang, S.; Scott, R. A.; Finnegan, M. G.; Johnson, M. K.; Ippolito, J. A.; Christianson, D. W.; Hausinger, R. P.

1993-01-01

206

A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis  

PubMed Central

Background Lactococcus lactis is widely used as a dairy starter and has been extensively studied. Based on the acquired knowledge on its physiology and metabolism, new applications have been envisaged and there is an increasing interest of using L. lactis as a cell factory. Plasmids constitute the main toolbox for L. lactis genetic engineering and most rely on antibiotic resistant markers for plasmid selection and maintenance. In this work, we have assessed the ability of the bacteriocin Lactococcin 972 (Lcn972) gene cluster to behave as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics. Lcn972 is a non-lantibiotic bacteriocin encoded by the 11-kbp plasmid pBL1 with a potent antimicrobial activity against Lactococcus. Results Attempts to clone the full lcn972 operon with its own promoter (P972), the structural gene lcn972 and the immunity genes orf2-orf3 in the unstable plasmid pIL252 failed and only plasmids with a mutated promoter were recovered. Alternatively, cloning under other constitutive promoters was approached and achieved, but bacteriocin production levels were lower than those provided by pBL1. Segregational stability studies revealed that the recombinant plasmids that yielded high bacteriocin titers were maintained for at least 200 generations without antibiotic selection. In the case of expression vectors such as pTRL1, the Lcn972 gene cluster also contributed to plasmid maintenance without compromising the production of the fluorescent mCherry protein. Furthermore, unstable Lcn972 recombinant plasmids became integrated into the chromosome through the activity of insertion sequences, supporting the notion that Lcn972 does apply a strong selective pressure against susceptible cells. Despite of it, the Lcn972 gene cluster was not enough to avoid the use of antibiotics to select plasmid-bearing cells right after transformation. Conclusions Inserting the Lcn972 cluster into segregational unstable plasmids prevents their lost by segregation and probable could be applied as an alternative to the use of antibiotics to support safer and more sustainable biotechnological applications of genetically engineered L. lactis. PMID:24886591

2014-01-01

207

Genome-scale genotype-phenotype matching of two Lactococcus lactis isolates from plants identifies mechanisms of adaptation to the plant niche  

Microsoft Academic Search

Lactococcus lactis is a primary constituent of many starter cultures used for the manufacturing of fermented dairy products, but the species also occurs in various nondairy niches such as (fermented) plant material. Three genome sequences of L. lactis dairy strains (IL-1403, SK11, and MG1363) are publicly available. An extensive molecular and phenotypic diversity analysis was now performed on two L.

Roland J. Siezen; Marjo J. C. Starrenburg; Jos Boekhorst; Bernadet Renckens; Douwe Molenaar

2008-01-01

208

Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling.  

PubMed

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from ?S1-casein and ?-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region. PMID:25465631

Brsting, M W; Qvist, K B; Brockmann, E; Vindelv, J; Pedersen, T L; Vogensen, F K; Ard, Y

2015-01-01

209

Study of the influence of yeast inoculum concentration (Yarrowia lipolytica and Kluyveromyces lactis) on blue cheese aroma development using microbiological models.  

PubMed

Yarrowia lipolytica and Kluyveromyces lactis occur as part of Stilton cheese microflora yet are not controlled during production. This study investigated the influence of their inoculum concentration on aroma production. Models of Y. lipolytica and K. lactis, with Penicillium roqueforti, were analysed using instrumental and sensory analysis. Different concentrations of Y. lipolytica produced important changes in the aroma profiles of microbiological models, analysed by solid-phase microextraction (SPME GC-MS). Sensory analysis with discrimination tests showed differences were detectable via human perception but did not concern the similarity to blue cheese odour. Increasing the inoculum concentration of K. lactis resulted in decreased variation between replicates. Partial least squares (PLS) regression on Flash profile data showed models inoculated with low concentrations of K. lactis exhibited blue cheese-related attributes, associated with increased ketone production. Results suggest that controlling the amount of Y. lipolytica and K. lactis during production offers potential to manipulate blue cheese aroma development. PMID:24128502

Price, Elliott J; Linforth, Robert S T; Dodd, Christine E R; Phillips, Carol A; Hewson, Louise; Hort, Joanne; Gkatzionis, Konstantinos

2014-02-15

210

Anti-inflammatory effects of Lactococcus lactis NCDO 2118 during the remission period of chemically induced colitis  

PubMed Central

Background Many probiotic bacteria have been described as promising tools for the treatment and prevention of inflammatory bowel diseases (IBDs). Most of these bacteria are lactic acid bacteria, which are part of the healthy human microbiota. However, little is known about the effects of transient bacteria present in normal diets, including Lactococcus lactis. Methods In the present study, we analysed the immunomodulatory effects of three L. lactis strains in vitro using intestinal epithelial cells. L. lactis NCDO 2118 was administered for 4days to C57BL/6 mice during the remission period of colitis induced by dextran sodium sulphate (DSS). Results Only one strain, L. lactis NCDO 2118, was able to reduce IL-1?-induced IL-8 secretion in Caco-2 cells, suggesting a potential anti-inflammatory effect. Oral treatment using L. lactis NCDO 2118 resulted in a milder form of recurrent colitis than that observed in control diseased mice. This protective effect was not attributable to changes in secretory IgA (sIgA); however, NCDO 2118 administration was associated with an early increase in IL-6 production and sustained IL-10 production in colonic tissue. Mice fed L. lactis NCDO 2118 had an increased number of regulatory CD4+ T cells (Tregs) bearing surface TGF-? in its latent form (Latency-associated peptide-LAP) in the mesenteric lymph nodes and spleen. Conclusions Here, we identified a new probiotic strain with a potential role in the treatment of IBD, and we elucidated some of the mechanisms underlying its anti-inflammatory effect. PMID:25110521

2014-01-01

211

Production of fermented milk using a malty compound-producing strain of Lactococcus lactis subsp. lactis biovar. diacetylactis, isolated from Zimbabwean naturally fermented milk.  

PubMed

Malty compound-producing Lactococcus lactis subsp. lactis biovar. diacetylactis strain INF-DM1, originally isolated from naturally fermented milk in Zimbabwe was used to prepare fermented milk from ordinary milk, milk enriched with 2.5% (w/v) skimmed milk powder and by 2.5% (w/v) increase in dry matter by ultrafiltration. Inoculated milk was incubated at 22, 30 and 37 degrees C. Analyses were made after 0, 9, 18 and 24 h incubation and also after 24 h incubation followed by storage for one week at 4 degrees C. Samples were analysed for volatile compounds including malty compounds and for organic acids, pH and log cfu/g. All samples were also judged for sensory attributes. Products made from enriched milks showed increased viscosity which was most marked in ultrafiltrated milk incubated at 30 and 37 degrees C. The levels of certain compounds (lactic acid, citrate and diacetyl) were significantly affected by milk type. Incubation temperature had a significant effect on starter growth rate and the rate of production and amount of the malty compounds, lactate, diacetyl, ethanol, acetoin and acetaldehyde. 3-Methyl butanal concentrations were above the taste threshold level of 0.06 ppm in almost all products, including stored products. Although initial growth rate was fastest at 37 degrees C, an uncoupling of acid production and growth was observed after 9 h incubation, suggesting that this is above the optimum temperature for this strain. In addition, products incubated at 37 degrees C showed a tendency to whey separation, indicating that this temperature is also too high to give optimum product quality. All products attained good scores in sensory analysis provided that fermentation was complete. Variation in the levels of malty compounds during the fermentation had no significant effect on the sensory score for total flavour. PMID:9631339

Narvhus, J A; Osteraas, K; Mutukumira, T; Abrahamsen, R K

1998-05-01

212

A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp. lactis BGMN1-5  

PubMed Central

Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors. PMID:24123824

Uzelac, Gordana; Lozo, Jelena; Aleksandrzak-Piekarczyk, Tamara; Gabrielsen, Christina; Kristensen, Tom; Nes, Ingolf F.; Diep, Dzung B.; Topisirovic, Ljubisa

2013-01-01

213

Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000  

PubMed Central

Background Listeria monocytogenes is a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactis NZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter. Results The constructed vector (pNZPnisA:CYTO-LLO) was expressed in L. lactis NZ9000 and was best induced at mid-log phase with 0.2% v/v nisin for 4 h statically at 30C. Purification of the His-tagged LLO was accomplished by Ni-NTA affinity chromatography and functionality was confirmed through haemolytic assays. Total LLO yield (measured as total protein content) was 4.435.9 mg per litre culture and the haemolytic activity was still detectable after 8 months of storage at 4C. Conclusion The LLO production method described in this work provides an approach to efficient LLO production in the Gram-positive Lactococcus bacterium to yield a significant source of the protein for research and diagnostic applications. Expression of LLO in L. lactis has a number of benefits over E. coli which may facilitate both in vivo and in vitro applications of this system. PMID:18664263

Bahey-El-Din, Mohammed; Griffin, Brendan T; Gahan, Cormac GM

2008-01-01

214

Optimization of fed-batch production of the model recombinant protein GFP in Lactococcus lactis.  

PubMed

Optimization of recombinant protein production using lactic acid bacteria (LAB) remains an important obstacle on the road to realizing LAB as oral vaccine delivery vehicles. Despite this, there have been few published investigations to explore the higher limits of LAB recombinant protein expression in fed-batch fermentations. In this study, results from response surface experiments suggested an optimal set of conditions for expression of green fluorescent protein (GFP), a model recombinant protein, in bench-scale, fed-batch Lactococcus lactis IL1403 fermentations. The 48 4-L fed-batch fermentations in this set of experiments, along with preliminary studies, investigated the effects of pH, temperature, hemin concentration, concentration of the nisin inducer per cell, and time of induction. Cell densities in this data set ranged from 2.9 to 7.4 g/L and maximum GFP expression per cell ranged from 0.1 to 4.4 relative fluorescence units (RFU)/g. The optimal 4-L, fed-batch fermentation process found here yields growth and protein expression values that dramatically improve upon results from traditional test tube and flask processes. Relative to the traditional process, the experimental optimum conditions yield 4.9 times the cell density, 1.6 times the protein per cell mass, and 8 times the total protein concentration. Unexpectedly, experiments also revealed that the compound hemin, known previously to improve growth and survival of Lactococcus lactis (L. lactis), negatively impacted recombinant protein production when added in concentrations from 5 to 20 microg/mL with this strain. The improvement in protein expression over traditional processes demonstrated here is an important step toward commercial development of LAB for oral delivery of recombinant vaccines and therapeutic proteins. PMID:17117427

Oddone, Gian M; Lan, Christopher Q; Rawsthorne, Helen; Mills, David A; Block, David E

2007-04-15

215

13C Nuclear Magnetic Resonance Studies of Citrate and Glucose Cometabolism by Lactococcus lactis  

PubMed Central

13C nuclear magnetic resonance (13C-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongrowing cells of Lactococcus lactis subsp. lactis 19B under anaerobic conditions. The metabolism of citrate plus glucose during growth was also monitored directly by in vivo NMR. Although pyruvate is a common intermediate metabolite in the metabolic pathways of both citrate and glucose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4-13C]citrate, [3-13C]pyruvate, and [2-13C]glucose. The presence of an additional substrate caused a considerable stimulation in the rates of substrate utilization, and the pattern of end products was changed. Acetate plus acetoin and butanediol represented more than 80% (molar basis) of the end products of the metabolism of citrate (or pyruvate) alone, but when glucose was also added, 80% of the citrate (or pyruvate) was converted to lactate. This result can be explained by the activation of lactate dehydrogenase by fructose 1,6-bisphosphate, an intermediate in glucose metabolism. The effect of different concentrations of glucose on the metabolism of citrate by dilute cell suspensions was also probed by using analytical methods other than NMR. Pyruvate dehydrogenase (but not pyruvate formate-lyase) was active in the conversion of pyruvate to acetyl coenzyme A. ?-Acetolactate was detected as an intermediate metabolite of citrate or pyruvate metabolism, and the labeling pattern of the end products agrees with the ?-acetolactate pathway. It was demonstrated that the contribution of the acetyl coenzyme A pathway for the synthesis of diacetyl, should it exist, is lower than 10%. Evidence for the presence of internal carbon reserves in L. lactis is presented. PMID:16349269

Ramos, Ana; Jordan, Kieran N.; Cogan, Timothy M.; Santos, Helena

1994-01-01

216

Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis.  

PubMed

Brazzein is an intensely sweet-tasting protein with high water solubility, heat stability, and taste properties resembling those of carbohydrate sweeteners. In the present study, we describe the expression of the synthetic gene encoding brazzein, a sweet protein in the yeast Kluyveromyces lactis. The synthetic brazzein gene was designed based on the biased codons of the yeast, so as to optimize its expression, as well as on the extracellular secretion for expression in an active, soluble form. The synthesized brazzein gene was cloned into the secretion vector pKLAC2, which contains the yeast prepropeptide signal from the Saccharomycescerevisiae ?-mating factor. The constructed plasmid pKLAC2-des-pE1M-brazzein was introduced into the yeast K. lactis GG799. The yeast transformants were cultured for high-yield secretion of the recombinant des-pE1M-brazzein in YPGal medium for 96 h at 30C. The expressed recombinant des-pE1M-brazzein was purified by CM-Sepharose column chromatography and approximately 104 mg/L was obtained. The purity and conformational state of the recombinant des-pE1M-brazzein were confirmed using SDS-PAGE, HPLC, and circular dichroism. The identity of the recombinant protein was also confirmed by N-terminal amino acid analysis and taste testing. The purified recombinant des-pE1M-brazzein had an intrinsic sweetness in its minor form, approximately 2130 times sweeter than sucrose on a weight basis. These results demonstrate that the K. lactis expression system is useful for producing the recombinant brazzein in active form at a high yield with attributes useful in the food industry. PMID:23684772

Jo, Hyun-Joo; Noh, Jin-Seok; Kong, Kwang-Hoon

2013-08-01

217

Analysis of the regions coding for transfer RNAs in Kluyveromyces lactis mitochondrial DNA.  

PubMed Central

The major regions coding for the transfer RNA genes in the mitochondrial DNA of K. lactis were studied. Twenty one, out of a supposed twenty four tRNA genes were identified and localized with respect to other mitochondrial genes. Most of the tRNA genes were found in a cluster downstream of the large ribosomal RNA gene. The order of a few groups of genes is conserved with respect to S. cerevisiae and T. glabrata. The highly diverged intergenic sequences contained a large number of guanine-cytosine clusters which frequently formed long palindromic sequences. PMID:2748331

Wilson, C; Ragnini, A; Fukuhara, H

1989-01-01

218

Crystallization and preliminary X-ray crystallographic analysis of ?-galactosidase from Kluyveromyces lactis  

PubMed Central

?-Galactosidase from Kluyveromyces lactis catalyses the hydrolysis of the ?-galactosidic linkage in lactose. Owing to its many industrial applications, the biotechnological potential of this enzyme is substantial. This protein has been expressed in yeast and purified for crystallization trials. However, optimization of the best crystallization conditions yielded crystals with poor diffraction quality that precluded further structural studies. Finally, the crystal quality was improved using the streak-seeding technique and a complete diffraction data set was collected at 2.8? resolution. PMID:20208165

Pereira-Rodrguez, ngel; Fernndez-Leiro, Rafael; Gonzlez Siso, M. Isabel; Cerdn, M. Esperanza; Becerra, Manuel; Sanz-Aparicio, Julia

2010-01-01

219

Characterization of the CopR regulon of Lactococcus lactis IL1403.  

PubMed

To identify components of the copper homeostatic mechanism of Lactococcus lactis, we employed two-dimensional gel electrophoresis to detect changes in the proteome in response to copper. Three proteins upregulated by copper were identified: glyoxylase I (YaiA), a nitroreductase (YtjD), and lactate oxidase (LctO). The promoter regions of these genes feature cop boxes of consensus TACAnnTGTA, which are the binding site of CopY-type copper-responsive repressors. A genome-wide search for cop boxes revealed 28 such sequence motifs. They were tested by electrophoretic mobility shift assays for the interaction with purified CopR, the CopY-type repressor of L. lactis. Seven of the cop boxes interacted with CopR in a copper-sensitive manner. They were present in the promoter region of five genes, lctO, ytjD, copB, ydiD, and yahC; and two polycistronic operons, yahCD-yaiAB and copRZA. Induction of these genes by copper was confirmed by real-time quantitative PCR. The copRZA operon encodes the CopR repressor of the regulon; a copper chaperone, CopZ; and a putative copper ATPase, CopA. When expressed in Escherichia coli, the copRZA operon conferred copper resistance, suggesting that it functions in copper export from the cytoplasm. Other member genes of the CopR regulon may similarly be involved in copper metabolism. PMID:17993525

Magnani, David; Barr, Olivier; Gerber, Simon D; Solioz, Marc

2008-01-01

220

Characterization of the mature cell surface proteinase of Lactobacillus delbrueckii subsp. lactis CRL 581.  

PubMed

The cell envelope-associated proteinase (CEP) of Lactobacillus delbrueckii subsp. lactis CRL 581 (PrtL) has an essential role in bacterial growth, contributes to the flavor and texture development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. The genome of L. delbrueckii subsp. lactis CRL 581 possesses only one gene that encodes PrtL, which consists of 1924 amino acids and is a multidomain protein anchored to the cell via its W domain. PrtL was extracted from the cell under high ionic strength conditions using NaCl, suggesting an electrostatic interaction between the proteinase and the cell envelope. The released PrtL was purified and biochemically characterized; its activity was maximal at temperatures between 37 and 40C and at pH between 7 and 8. Under optimal conditions, PrtL exhibited higher affinity for succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide than for succinyl-alanyl-glutamyl-prolyl-phenylalanine-p-nitroanilide, while methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide was not degraded. A similar ?- and ?-casein degradation pattern was observed with the purified and the cell envelope-bound proteinase. Finally, on the basis of its specificity towards caseins and the unique combination of amino acids at residues thought to be involved in substrate specificity, PrtL can be classified as a representative of a new group of CEP. PMID:25487890

Villegas, Josefina M; Brown, Luca; Savoy de Giori, Graciela; Hebert, Elvira M

2014-12-01

221

Lactose-induced cell death of beta-galactosidase mutants in Kluyveromyces lactis.  

PubMed

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches. PMID:15851101

Lodi, Tiziana; Donnini, Claudia

2005-05-01

222

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1.  

PubMed

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium. PMID:17156020

Baruffini, Enrico; Goffrini, Paola; Donnini, Claudia; Lodi, Tiziana

2006-12-01

223

Multilocus Sequence Typing Scheme for the Characterization of 936-Like Phages Infecting Lactococcus lactis  

PubMed Central

Lactococcus lactis phage infections are costly for the dairy industry because they can slow down the fermentation process and adversely impact product safety and quality. Although many strategies have been developed to better control phage populations, new virulent phages continue to emerge. Thus, it is beneficial to develop an efficient method for the routine identification of new phages within a dairy plant to rapidly adapt antiphage tactics. Here, we present a multilocus sequence typing (MLST) scheme for the characterization of the 936-like phages, the most prevalent phage group infecting L. lactis strains worldwide. The proposed MLST system targets the internal portion of five highly conserved genomic sequences belonging to the packaging, morphogenesis, and lysis modules. Our MLST scheme was used to analyze 100 phages with different restriction fragment length polymorphism (RFLP) patterns isolated from 11 different countries between 1971 and 2010. PCR products were obtained for all the phages analyzed, and sequence analysis highlighted the high discriminatory power of the MLST system, detecting 93 different sequence types. A conserved locus within the lys gene (coding for endolysin) was the most discriminative, with 65 distinct alleles. The locus within the mcp gene (major capsid protein) was the most conserved (54 distinct alleles). Phylogenetic analyses of the concatenated sequences exhibited a strong concordance of the clusters with the phage host range, indicating the clonal evolution of these phages. A public database has been set up for the proposed MLST system, and it can be accessed at http://pubmlst.org/bacteriophages/. PMID:22522686

Moisan, Maxim

2012-01-01

224

Molecular Characterization of a Recombinant Manganese Superoxide Dismutase from Lactococcus lactis M4  

PubMed Central

A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967?U/mg when induced with 1?mM of isopropyl-?-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27?kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25C and pH 7.2. It was stable up to 45C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172). PMID:24592392

Chor Leow, Thean; Foo, Hooi Ling; Abdul Rahim, Raha

2014-01-01

225

The Carbohydrate Metabolism Signature of Lactococcus lactis Strain A12 Reveals Its Sourdough Ecosystem Origin  

PubMed Central

Lactococcus lactis subsp. lactis strain A12 was isolated from sourdough. Combined genomic, transcriptomic, and phenotypic analyses were performed to understand its survival capacity in the complex sourdough ecosystem and its role in the microbial community. The genome sequence comparison of strain A12 with strain IL1403 (a derivative of an industrial dairy strain) revealed 78 strain-specific regions representing 23% of the total genome size. Most of the strain-specific genes were involved in carbohydrate metabolism and are potentially required for its persistence in sourdough. Phenotype microarray, growth tests, and analysis of glycoside hydrolase content showed that strain A12 fermented plant-derived carbohydrates, such as arabinose and ?-galactosides. Strain A12 exhibited specific growth rates on raffinose that were as high as they were on glucose and was able to release sucrose and galactose outside the cell, providing soluble carbohydrates for sourdough microflora. Transcriptomic analysis identified genes specifically induced during growth on raffinose and arabinose and reveals an alternative pathway for raffinose assimilation to that used by other lactococci. PMID:23872564

Passerini, Delphine; Coddeville, Michle; Le Bourgeois, Pascal; Loubire, Pascal; Ritzenthaler, Paul; Fontagn-Faucher, Catherine; Cocaign-Bousquet, Muriel

2013-01-01

226

Statistical investigation of Kluyveromyces lactis cells permeabilization with ethanol by response surface methodology  

PubMed Central

The aim of our study was to select the optimal operating conditions to permeabilize Kluyveromyces lactis cells using ethanol as a solvent as an alternative to cell disruption and extraction. Cell permeabilization was carried out by a non-mechanical method consisting of chemical treatment with ethanol, and the results were expressed as ?-galactosidase activity. Experiments were conducted under different conditions of ethanol concentration, treatment time and temperature according to a central composite rotatable design (CCRD), and the collected results were then worked out by response surface methodology (RSM). Cell permeabilization was improved by an increase in ethanol concentration and simultaneous decreases in the incubation temperature and treatment time. Such an approach allowed us to identify an optimal range of the independent variables within which the ?-galactosidase activity was optimized. A maximum permeabilization of 2,816 mmol L?1 oNP min?1 g?1 was obtained by treating cells with 75.0% v/v of ethanol at 20.0 C for 15.0 min. The proposed methodology resulted to be effective and suited for K. lactis cells permeabilization at a lab-scale and promises to be of possible interest for future applications mainly in the food industry. PMID:24688494

de Faria, Janana T.; Rocha, Pollyana F.; Converti, Attilio; Passos, Flvia M.L.; Minim, Luis A.; Sampaio, Fbio C.

2013-01-01

227

Influence of growth conditions on the nisin production of bioengineered Lactococcus lactis strains.  

PubMed

Nisin production of three bioengineered strains, (LAC338, LAC339 and LAC340) with immunity (nisFEG) and/or regulation (nisRK) genes of nisin biosynthesis on plasmids in the Lactococcus lactis LL27 nisin producer, was evaluated under pH-controlled and pH-uncontrolled batch fermentations. Optimization studies showed that fructose and yeast extract yielded the highest nisin activity. The strains LAC338, LAC339, and LAC340 produced 24, 45, and 44% more nisin, respectively, than wild-type L. lactis LL27 after 12-h incubation. However, sharp decreases in the yield of nisin were observed at the late phase of fermentation with LAC339 and LL27 in contrast to LAC340 and LAC338 strains for which the high level of nisin could be maintained longer. Obviously, increasing the copy number of the regulation genes together with immunity genes in the nisin producers retarded the loss of nisin in the late phase of the fermentation. PMID:19137338

Sim?ek, O; Con, A H; Akko, N; Saris, P E J; Akelik, Mustafa

2009-04-01

228

Purification and characterization of a novel glucansucrase from Leuconostoc lactis EG001.  

PubMed

A gene encoding glucansucrase was identified in Leuconostoc lactis EG001 isolated from lactic acid bacteria (LAB) in Kimchi, a traditional Korean fermented food. The L. lactis EG001 glucansucrase gene consists of 4503 bp open reading frame (ORF) and encodes an enzyme of 1500 amino acids with an apparent molecular mass of 165 kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity (75%) to that of dextransucrase of L. mesenteroides. The gene was cloned and over-expressed in Escherichia coli strain. The recombinant enzyme was purified via Ni-NTA affinity chromatography and its enzymatic properties were characterized. The enzyme exhibited optimum activity at 30 degrees C and pH 5.0. In addition, the enzyme was able to catalyze the glycosylation of l-ascorbic acid to l-ascorbic acid 2-glucoside. The glycosylated product via EG001 glucansucrase has the potential as an antioxidant in industrial applications. PMID:19853426

Kim, Yong-Mo; Yeon, Min Ji; Choi, Nack-Shick; Chang, Young-Hyo; Jung, Min Young; Song, Jae Jun; Kim, Joong Su

2010-07-20

229

Signals of aging associated with lower growth rates in Kluyveromyces lactis cultures under nitrogen limitation.  

PubMed

The effects of aging on the specific growth rate of Kluyveromyces lactis cultures, as a function of (NH4)2SO4 concentration, were evaluated. The growth kinetic parameters maximum specific growth rate and saturation constant for (NH4)2SO4 were calculated to be 0.44 h(-1) and 0.15 mmolL(-1), respectively. Batch cultures were allowed to age for 16 days without influence of cell density or starvation. The specific growth rates of these cultures were determined each day and decreased as the population aged at different nitrogen concentrations. Aging signals (N-acetylglucosamine content of the cell wall, cell dimensions, and apoptosis markers) were measured. Apoptosis markers were detected after 5 days at limiting (NH4)2SO4 concentrations (0.57, 3.80, and 7.60 mmolL(-1)) but only after 8 days at a nonlimiting (NH4)2SO4 concentration (38.0 mmolL(-1)). Similarly, continuous cultures of K. lactis performed under nitrogen limitation and, at lower dilution rates, accumulated cells exhibiting aging signals. The results demonstrate that aging affects growth rate and raise the question of whether nitrogen limitation accelerates aging. Because aging is correlated with growth rate, and each dilution rate of the continuous cultures tends to select and accumulate cells with a respective age, cultures growing at lower growth rates can be useful to investigate yeast physiological responses, including aging. PMID:25204685

Corra, Lygia Ftima da Mata; Passos, Frederico Jos Vieira; Viloria, Marlene Isabel Vargas; Martins Filho, Olindo Assis; de Carvalho, Andra Teixeira; Passos, Flvia Maria Lopes

2014-09-01

230

Contribution of Lactococcus lactis reducing properties to the downregulation of a major virulence regulator in Staphylococcus aureus, the agr system.  

PubMed

Staphylococcus aureus is a major cause of food poisoning outbreaks associated with dairy products, because of the ingestion of preformed enterotoxins. The biocontrol of S. aureus using lactic acid bacteria (LAB) offers a promising opportunity to fight this pathogen while respecting the product ecosystem. We had previously established the ability of Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, to downregulate a major staphylococcal virulence regulator, the accessory gene regulator (agr) system, and, as a consequence, agr-controlled enterotoxins. In the present paper, we have shown that the oxygen-independent reducing properties of L. lactis contribute to agr downregulation. Neutralizing lactococcal reduction by adding potassium ferricyanide or maintaining the oxygen pressure constant at 50% released agr downregulation in the presence of L. lactis. This downregulation still occurred in an S. aureus srrA mutant, indicating that the staphylococcal respiratory response regulator SrrAB was not the only component in the signaling pathway. Therefore, this study clearly demonstrates the ability of L. lactis reducing properties to interfere with the expression of S. aureus virulence, thus highlighting this general property of LAB as a lever to control the virulence expression of this major pathogen in a food context and beyond. PMID:25192992

Nouaille, Sbastien; Rault, Lucie; Jeanson, Sophie; Loubire, Pascal; Le Loir, Yves; Even, Sergine

2014-11-01

231

Comparison of the effects of various attenuation methods on cell permeability and accessibility of intracellular enzymes in Lactococcus lactis strains  

Microsoft Academic Search

Three Lactococcus lactis cheese starter cultures were exposed to various cell attenuation methods including sonication, chemical treatments, heat-shock and freeze-shock. Treated cells were subsequently assessed for changes in cell permeability and accessibility to the intracellular peptidases Pep X and Pep N. Generally, for all three strains, as the duration of sonication increased, the levels of intracellular peptidases in the cell

Imelda A. Doolan; Martin G. Wilkinson

2009-01-01

232

Effect of autochthonous bacteriocin-producing Lactococcus lactis on bacterial population dynamics and growth of halotolerant bacteria in Brazilian charqui.  

PubMed

Charqui is a fermented, salted and sun-dried meat product, widely consumed in Brazil and exported to several countries. Growth of microorganisms in this product is unlikely due to reduced Aw, but halophilic and halotolerant bacteria may grow and cause spoilage. Charqui is a good source of lactic acid bacteria able to produce antimicrobial bacteriocins. In this study, an autochthonous bacteriocinogenic strain (Lactococcus lactis subsp. lactis 69), isolated from charqui, was added to the meat used for charqui manufacture and evaluated for its capability to prevent the growth of spoilage bacteria during storage up to 45 days. The influence of L.lactis 69 on the bacterial diversity during the manufacturing of the product was also studied, using denaturing gradient gel electrophoresis (DGGE). L.lactis 69 did not affect the counts and diversity of lactic acid bacteria during manufacturing and storage, but influenced negatively the populations of halotolerant microorganisms, reducing the spoilage potential. The majority of tested virulence genes was absent, evidencing the safety and potential technological application of this strain as an additional hurdle to inhibit undesirable microbial growth in this and similar fermented meat products. PMID:25084676

Biscola, Vanessa; Abriouel, Hikmate; Todorov, Svetoslav Dimitrov; Capuano, Verena Sant'Anna Cabral; Glvez, Antonio; Franco, Bernadette Dora Gombossy de Melo

2014-12-01

233

Assessing the effects of exposure to environmental stress on some functional properties of Bifidobacterium animalis ssp. lactis.  

PubMed

This study assessed the effects of exposing a strain of Bifidobacterium animalis ssp. lactis to acid, bile and osmotic stresses on antagonistic properties, biofilm formation and antibiotic susceptibility/resistance profile. Exposure to each stress factor appeared to have no significant effect on the antagonism against Escherichia coli NCTC 12900 and Salmonella enterica serovar Enteritidis PT4. No suppression in biofilm formation due to exposure to stress was observed. Bile and osmotic stresses resulted in significantly higher biofilm formation. Expression of an exopolysaccharide synthesis gene, gtf 01207, was significantly higher when the B. animalis ssp. lactis strain was exposed to osmotic stress. Susceptibility of the B. animalis ssp. lactis strain to chloramphenicol, erythromycin, ampicillin and vancomycin, and resistance to tetracycline remained unchanged when exposed to each stress. The expression of a tetracycline resistance gene, tet(W), was significantly higher when exposed to each stress. These results may suggest that the potential for the B. animalis ssp. lactis strain to provide probiotic benefit, after exposure to the stressful conditions of the gastrointestinal tract, remains intact. PMID:25097108

Amund, O D; Ouoba, L I I; Sutherland, J P; Ghoddusi, H B

2014-12-01

234

Genotypic and Phenotypic Analysis of Dairy Lactococcus lactis Biodiversity in Milk: Volatile Organic Compounds as Discriminating Markers  

PubMed Central

The diversity of nine dairy strains of Lactococcus lactis subsp. lactis in fermented milk was investigated by both genotypic and phenotypic analyses. Pulsed-field gel electrophoresis and multilocus sequence typing were used to establish an integrated genotypic classification. This classification was coherent with discrimination of the L. lactis subsp. lactis bv. diacetylactis lineage and reflected clonal complex phylogeny and the uniqueness of the genomes of these strains. To assess phenotypic diversity, 82 variables were selected as important dairy features; they included physiological descriptors and the production of metabolites and volatile organic compounds (VOCs). Principal-component analysis (PCA) demonstrated the phenotypic uniqueness of each of these genetically closely related strains, allowing strain discrimination. A method of variable selection was developed to reduce the time-consuming experimentation. We therefore identified 20 variables, all associated with VOCs, as phenotypic markers allowing discrimination between strain groups. These markers are representative of the three metabolic pathways involved in flavor: lipolysis, proteolysis, and glycolysis. Despite great phenotypic diversity, the strains could be divided into four robust phenotypic clusters based on their metabolic orientations. Inclusion of genotypic diversity in addition to phenotypic characters in the classification led to five clusters rather than four being defined. However, genotypic characters make a smaller contribution than phenotypic variables (no genetic distances selected among the most contributory variables). This work proposes an original method for the phenotypic differentiation of closely related strains in milk and may be the first step toward a predictive classification for the manufacture of starters. PMID:23709512

Dhaisne, Amandine; Guellerin, Maeva; Laroute, Valrie; Laguerre, Sandrine; Le Bourgeois, Pascal; Loubiere, Pascal

2013-01-01

235

Spatial competition with Lactococcus lactis in mixed-species continuous-flow biofilms inhibits Listeria monocytogenes growth  

Microsoft Academic Search

Surfaces in industrial settings provide a home for resident biofilms that are likely to interact with the attachment, growth and survival of pathogens such as Listeria monocytogenes. Experimental results have indicated that L. monocytogenes cells were inhibited by the presence of a model resident flora (Lactococcus lactis) in dual-species continuous flow-biofilms, and are spatially restricted to the lower biofilm layers.

Olivier Habimana; Laurent Guillier; Saulius Kulakauskas; Romain Briandet

2011-01-01

236

Multilocus sequence typing of Lactococcus lactis from naturally fermented milk foods in ethnic minority areas of China.  

PubMed

To determine the genetic diversity and phylogenetic relationships among Lactococcus lactis isolates, 197 strains isolated from naturally homemade yogurt in 9 ethnic minority areas of 6 provinces of China were subjected to multilocus sequence typing (MLST). The MLST analysis was performed using internal fragment sequences of 12 housekeeping genes (carB, clpX, dnaA, groEL, murC, murE, pepN, pepX, pyrG, recA, rpoB, and pheS). Six (dnaA) to 8 (murC) different alleles were detected for these genes, which ranged from 33.62 (clpX) to 41.95% (recA) GC (guanine-cytosine) content. The nucleotide diversity (?) ranged from 0.00362 (murE) to 0.08439 (carB). Despite this limited allelic diversity, the allele combinations of each strain revealed 72 different sequence types, which denoted significant genotypic diversity. The dN/dS ratios (where dS is the number of synonymous substitutions per synonymous site, and dN is the number of nonsynonymous substitutions per nonsynonymous site) were lower than 1, suggesting potential negative selection for these genes. The standardized index of association of the alleles IA(S)=0.3038 supported the clonality of Lc. lactis, but the presence of network structure revealed by the split decomposition analysis of the concatenated sequence was strong evidence for intraspecies recombination. Therefore, this suggests that recombination contributed to the evolution of Lc. lactis. A minimum spanning tree analysis of the 197 isolates identified 14 clonal complexes and 23 singletons. Phylogenetic trees were constructed based on the sequence types, using the minimum evolution algorithm, and on the concatenated sequence (6,192 bp), using the unweighted pair-group method with arithmetic mean, and these trees indicated that the evolution of our Lc. lactis population was correlated with geographic origin. Taken together, our results demonstrated that MLST could provide a better understanding of Lc. lactis genome evolution, as well as useful information for future studies on global Lc. lactis structure and genetic evolution, which will lay the foundation for screening Lc. lactis as starter cultures in fermented dairy products. PMID:24612812

Xu, Haiyan; Sun, Zhihong; Liu, Wenjun; Yu, Jie; Song, Yuqin; Lv, Qiang; Zhang, Jiachao; Shao, Yuyu; Menghe, Bilige; Zhang, Heping

2014-05-01

237

Intra- and Interspecies Conjugal Transfer of Tn916-Like Elements from Lactococcus lactis In Vitro and In Vivo?  

PubMed Central

Tetracycline-resistant Lactococcus lactis strains originally isolated from Polish raw milk were analyzed for the ability to transfer their antibiotic resistance genes in vitro, using filter mating experiments, and in vivo, using germfree rats. Four of six analyzed L. lactis isolates were able to transfer tetracycline resistance determinants in vitro to L. lactis Bu2-60, at frequencies ranging from 10?5 to 10?7 transconjugants per recipient. Three of these four strains could also transfer resistance in vitro to Enterococcus faecalis JH2-2, whereas no transfer to Bacillus subtilis YBE01, Pseudomonas putida KT2442, Agrobacterium tumefaciens UBAPF2, or Escherichia coli JE2571 was observed. Rats were initially inoculated with the recipient E. faecalis strain JH2-2, and after a week, the L. lactis IBB477 and IBB487 donor strains were introduced. The first transconjugants were detected in fecal samples 3 days after introduction of the donors. A subtherapeutic concentration of tetracycline did not have any significant effect on the number of transconjugants, but transconjugants were observed earlier in animals dosed with this antibiotic. Molecular analysis of in vivo transconjugants containing the tet(M) gene showed that this gene was identical to tet(M) localized on the conjugative transposon Tn916. Primer-specific PCR confirmed that the Tn916 transposon was complete in all analyzed transconjugants and donors. This is the first study showing in vivo transfer of a Tn916-like antibiotic resistance transposon from L. lactis to E. faecalis. These data suggest that in certain cases food lactococci might be involved in the spread of antibiotic resistance genes to other lactic acid bacteria. PMID:19666731

Boguslawska, Joanna; Zycka-Krzesinska, Joanna; Wilcks, Andrea; Bardowski, Jacek

2009-01-01

238

Production of lactic acid from date juice extract with free cells of single and mixed cultures of Lactobacillus casei and Lactococcus lactis  

Microsoft Academic Search

The production of lactic acid from date juice by single and mixed cultures of Lactobacillus casei and Lactococcus lactis was investigated. In the present conditions, the highest concentration of lactic acid (60.3gl?1) was obtained in the mixed culture system while in single culture fermentations of Lactobacillus casei or Lactococcus lactis, the maximum concentration of lactic acid was 53 and 46gl?1,

Aicha Nancib; Nabil Nancib; Joseph Boudrant

2009-01-01

239

Effect of Sub-inhibitory Amounts of Nisin and Mineral Salts on Nisin Production by Lactococcus lactis UQ2 in Skim Milk  

Microsoft Academic Search

The effect of nisin regulatory system of the quorum-sensing mechanism and mineral salts on the production of nisin A by the\\u000a native strain Lactococcus lactis UQ2 growing in skim milk was evaluated using a static culture. A 6??3 full factorial design with two replicates was conducted,\\u000a aiming to study nisin production during growth of L. lactis UQ2 in skim milk

Mara D. Garca-Parra; Blanca E. Garca-Almendrez; Lorenzo Guevara-Olvera; Ramn G. Guevara-Gonzlez; Ana Rodrguez; Beatriz Martnez; Jorge Domnguez-Domnguez; Carlos Regalado

2011-01-01

240

Utilisation of galacto-oligosaccharides as selective substrates for growth by lactic acid bacteria including Bifidobacterium lactis DR10 and Lactobacillus rhamnosus DR20  

Microsoft Academic Search

Two probiotic strains of bacteria Bifidobacterium lactis DR10 and Lactobacillus rhamnosus DR20 were tested for their ability to utilise and grow on galacto-oligosaccharides present in a commercial hydrolysed lactose milk powder. The results clearly demonstrated that B. lactis DR10 preferentially utilises tri- and tetra-saccharides whereas Lb. rhamnosus DR20 prefers sugars with a lower degree of polymerisation, i.e., disaccharides and monosaccharides.

Pramod K Gopal; Patrick A Sullivan; John B Smart

2001-01-01

241

Improved homo l -lactic acid fermentation from xylose by abolishment of the phosphoketolase pathway and enhancement of the pentose phosphate pathway in genetically modified xylose-assimilating Lactococcus lactis  

Microsoft Academic Search

In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation\\u000a ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK

Satoru Shinkawa; Kenji Okano; Shogo Yoshida; Tsutomu Tanaka; Chiaki Ogino; Hideki Fukuda; Akihiko Kondo

242

Cell Surface of Lactococcus lactis Is Covered by a Protective Polysaccharide Pellicle*  

PubMed Central

In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria. PMID:20106971

Chapot-Chartier, Marie-Pierre; Vinogradov, Evgeny; Sadovskaya, Irina; Andre, Guillaume; Mistou, Michel-Yves; Trieu-Cuot, Patrick; Furlan, Sylviane; Bidnenko, Elena; Courtin, Pascal; Pchoux, Christine; Hols, Pascal; Dufrne, Yves F.; Kulakauskas, Saulius

2010-01-01

243

Molecular Insights on the Recognition of a Lactococcus lactis Cell Wall Pellicle by the Phage 1358 Receptor Binding Protein  

PubMed Central

ABSTRACT The Gram-positive bacterium Lactococcus lactis is used for the production of cheeses and other fermented dairy products. Accidental infection of L. lactis cells by virulent lactococcal tailed phages is one of the major risks of fermentation failures in industrial dairy factories. Lactococcal phage 1358 possesses a host range limited to a few L. lactis strains and strong genomic similarities to Listeria phages. We report here the X-ray structures of phage 1358 receptor binding protein (RBP) in complex with monosaccharides. Each monomer of its trimeric RBP is formed of two domains: a shoulder domain linking the RBP to the rest of the phage and a jelly roll fold head/host recognition domain. This domain harbors a saccharide binding crevice located in the middle of a monomer. Crystal structures identified two sites at the RBP surface, ?8 ? from each other, one accommodating a GlcNAc monosaccharide and the other accommodating a GlcNAc or a glucose 1-phosphate (Glc1P) monosaccharide. GlcNAc and GlcNAc1P are components of the polysaccharide pellicle that we identified at the cell surface of L. lactis SMQ-388, the host of phage 1358. We therefore modeled a galactofuranose (Galf) sugar bridging the two GlcNAc saccharides, suggesting that the trisaccharidic motif GlcNAc-Galf-GlcNAc (or Glc1P) might be common to receptors of genetically distinct lactococcal phages p2, TP091-1, and 1358. Strain specificity might therefore be elicited by steric clashes induced by the remaining components of the pellicle hexasaccharide. Taken together, these results provide a first insight into the molecular mechanism of host receptor recognition by lactococcal phages. IMPORTANCE Siphophages infecting the Gram-positive bacterium Lactococcus lactis are sources of milk fermentation failures in the dairy industry. We report here the structure of the pellicle polysaccharide from L. lactis SMQ-388, the specific host strain of phage 1358. We determined the X-ray structures of the lytic lactococcal phage 1358 receptor binding protein (RBP) in complex with monosaccharides. The positions and nature of monosaccharides bound to the RBP are in agreement with the pellicle structure and suggest a general binding mode of lactococcal phages to their pellicle saccharidic receptor. PMID:24719416

Farenc, Carine; Spinelli, Silvia; Vinogradov, Evgeny; Tremblay, Denise; Blangy, Stphanie; Sadovskaya, Irina

2014-01-01

244

Biosynthesis and stereochemical configuration of N5-(1-carboxyethyl)ornithine. An unusual amino acid produced by Streptococcus lactis  

SciTech Connect

In a recent communication we described the purification and characterization of N5-(1-carboxyethyl)ornithine from cells of Streptococcus lactis 133. This unusual amino acid has not previously been found in nature. Radiotracer experiments presented here reveal that exogenous (/sup 14/C)ornithine serves as the precursor for biosynthesis of (/sup 14/C)arginine, (/sup 14/C)N5-(1-carboxyethyl)ornithine, and (/sup 14/C)N5-acetylornithine by cells of S. lactis K1 during growth in a defined medium lacking arginine. In the absence of both arginine and ornithine, cells of S. lactis K1 can also generate intracellular (/sup 14/C)N5-(1-carboxyethyl)ornithine from exogenous (/sup 14/C)glutamic acid. Previously we showed that the properties of N5-(1-carboxyethyl)ornithine prepared from S. lactis were identical to one of the two diastereomers (2S, 7S) or (2S, 7R) present in a synthetic preparation of (2S, 7RS)-N5-(1-carboxyethyl)ornithine. The two diastereomers have now been unambiguously synthesized by an Abderhalden-Haase condensation between (2S)-N2-t-butoxycarbonyl-ornithine and the chiral (2S)-, and (2R)-bromopropionates. By /sup 13/C-NMR spectroscopy it has been established that the preparation from S. lactis is exclusively (2S, 7S)-N5-(1-carboxyethyl)ornithine. has been demonstrated in a cell-free extract of S. lactis 133. The requirements for ornithine, pyruvic acid, and NAD(P)H suggest that biosynthesis of N5-(1-carboxyethyl)ornithine occurs via a reductive condensation mechanism. A general survey revealed that N5-(1-carboxyethyl)ornithine was produced only by certain strains of Group N streptococci. These findings may indicate a plasmid locus for the gene(s) encoding the enzyme(s) for N5-(1-carboxyethyl)ornithine biosynthesis.

Miller, S.P.; Thompson, J.

1987-11-25

245

Rerouting Citrate Metabolism in Lactococcus lactis to Citrate-Driven Transamination  

PubMed Central

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:40494056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding ?-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both ?-keto acids and ?-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate. PMID:22798354

Pudlik, Agata M.

2012-01-01

246

How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis  

PubMed Central

Background In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus Trichoderma reesei. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis. Results After expression in the yeast Kluyveromyces lactis, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from T. reesei. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin-induced reduction in particle size and crystallinity resulted in high cellulose hydrolysis rates. Conclusions Recombinant swollenin can be easily produced with the robust yeast K. lactis. Moreover, swollenin induces deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization). For the first time, this study quantifies and elucidates in detail how swollenin affects different cellulosic substrates and their hydrolysis. PMID:21943248

2011-01-01

247

Structural Basis for the Transcriptional Regulation of Heme Homeostasis in Lactococcus lactis*  

PubMed Central

Although heme is a crucial element for many biological processes including respiration, heme homeostasis should be regulated strictly due to the cytotoxicity of free heme molecules. Numerous lactic acid bacteria, including Lactococcus lactis, acquire heme molecules exogenously to establish an aerobic respiratory chain. A heme efflux system plays an important role for heme homeostasis to avoid cytotoxicity of acquired free heme, but its regulatory mechanism is not clear. Here, we report that the transcriptional regulator heme-regulated transporter regulator (HrtR) senses and binds a heme molecule as its physiological effector to regulate the expression of the heme-efflux system responsible for heme homeostasis in L. lactis. To elucidate the molecular mechanisms of how HrtR senses a heme molecule and regulates gene expression for the heme efflux system, we determined the crystal structures of the apo-HrtRDNA complex, apo-HrtR, and holo-HrtR at a resolution of 2.0, 3.1, and 1.9 ?, respectively. These structures revealed that HrtR is a member of the TetR family of transcriptional regulators. The residue pair Arg-46 and Tyr-50 plays a crucial role for specific DNA binding through hydrogen bonding and a CH-? interaction with the DNA bases. HrtR adopts a unique mechanism for its functional regulation upon heme sensing. Heme binding to HrtR causes a coil-to-helix transition of the ?4 helix in the heme-sensing domain, which triggers a structural change of HrtR, causing it to dissociate from the target DNA for derepression of the genes encoding the heme efflux system. HrtR uses a unique heme-sensing motif with bis-His (His-72 and His-149) ligation to the heme, which is essential for the coil-to-helix transition of the ?4 helix upon heme sensing. PMID:22798069

Sawai, Hitomi; Yamanaka, Masaru; Sugimoto, Hiroshi; Shiro, Yoshitsugu; Aono, Shigetoshi

2012-01-01

248

Milk Fermented with a 15-Lipoxygenase-1-Producing Lactococcus Lactis Alleviates Symptoms of colitis in a Murine Model.  

PubMed

Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, are characterized by extensive inflammation due to dysregulation of the innate and adaptive immune system whose exact etiology is not yet completely understood. Currently there is no cure for IBD, thus the search for new molecules capable of controlling IBD and their delivery to the site of inflammation are the goal of many researchers. The aim of this work was to evaluate the anti-inflammatory effect of the administration of milks fermented by a Lactococcus (L.) lactis strain producing 15-lipoxygenase-1 (15-LOX-1) using a trinitrobenzenesulfonic acid-induced IBD mouse model. The results obtained demonstrated that 15-LOX-1 producing L. lactis was effective in the prevention of the intestinal damage associated to inflammatory bowel disease in a murine model. The work also confirmed previous studies showing that fermented milk is an effective form of administration of recombinant lactic acid bacteria expressing beneficial molecules. PMID:25395213

Saraiva, Tesslia Diniz Luerce; Morais, Ktia; Pereira, Vanessa Bastos; de Azevedo, Marcela; Rocha, Clarissa Santos; Prosperi, Camila Castro; Gomes-Santos, Ana Cristina; Bermudez-Humaran, Luis; Faria, Ana Maria Caetano; Blottiere, Herv M; Langella, Philippe; Miyoshi, Anderson; de LeBlanc, Alejandra de Moreno; LeBlanc, Jean Guy; Azevedo, Vasco

2014-11-13

249

Characterization of growth and enzymes produced by prt+lac+ and prt-lac- Lactococcus lactis cells  

Microsoft Academic Search

Lactococcus lactis subsp.cremoris strains KH (prt+lac+) and KHA (prt?lac?) are used as starter cultures in manufacture of ripened cheeses. The growth characteristics and the dipeptidases produced\\u000a by these cells have been investigated in this study. A new semisynthetic medium was developed for growth of the cells. Growth\\u000a on this medium was as good as or better than that on milk,

Joong Kim; Rakesh Bajpai; Robert T. Marshall

1993-01-01

250

Perdeuteration and methyl-selective (1)H, (13)C-labeling by using a Kluyveromyces lactis expression system.  

PubMed

The production of stable isotope-labeled proteins is critical in structural analyses of large molecular weight proteins using NMR. Although prokaryotic expression systems using Escherichia coli have been widely used for this purpose, yeast strains have also been useful for the expression of functional eukaryotic proteins. Recently, we reported a cost-effective stable isotope-labeled protein expression using the hemiascomycete yeast Kluyveromyces lactis (K. lactis), which allow us to express exogenous proteins at costs comparable to prokaryotic expression systems. Here, we report the successful production of highly deuterated (>90 %) protein in the K. lactis system. We also examined the methyl-selective (1)H, (13)C-labeling of Ile, Leu, and Val residues using commonly used amino acid precursors. The efficiency of (1)H-(13)C-incorporation varied significantly based on the amino acid. Although a high level of (1)H-(13)C-incorporation was observed for the Ile ?1 position, (1)H, (13)C-labeling rates of Val and Leu methyl groups were limited due to the mitochondrial localization of enzymes involved in amino acid biosynthesis and the lack of transporters for ?-ketoisovalerate in the mitochondrial membrane. In line with this notion, the co-expression with branched-chain-amino-acid aminotransferase in the cytosol significantly improved the incorporation rates of amino acid precursors. Although it would be less cost-effective, addition of (13)C-labeled valine can circumvent problems associated with precursors and achieve high level (1)H, (13)C-labeling of Val and Leu. Taken together, the K. lactis system would be a good alternative for expressing large eukaryotic proteins that need deuteration and/or the methyl-selective (1)H, (13)C-labeling for the sensitive detection of NMR resonances. PMID:24146206

Miyazawa-Onami, Mayumi; Takeuchi, Koh; Takano, Toshiaki; Sugiki, Toshihiko; Shimada, Ichio; Takahashi, Hideo

2013-11-01

251

Kluyveromyces lactis Sir2p regulates cation sensitivity and maintains a specialized chromatin structure at the cryptic alpha-locus.  

PubMed Central

In Saccharomyces cerevisiae, transcriptional silencing of the cryptic mating type loci requires the formation of a heterochromatin-like structure, which is dependent on silent information regulator (Sir) proteins and DNA sequences, called silencers. To learn more about silencing, we characterized the mating type loci from the yeast Kluyveromyces lactis. The K. lactis MAT, HMRa, and HMLalpha loci shared flanking DNA sequences on both sides of the loci presumably acting as recombinational targets during mating type switching. HMRa contained two genes, the a1 gene similar to the Saccharomyces a1 gene and the a2 gene similar to mating type genes from other yeasts. K. lactis HMLalpha contained three genes, the alpha1 and alpha2 genes, which were similar to their Saccharomyces counterparts, and a novel third gene, alpha3. A dam-methylase assay showed Sir-dependent, but transcription-independent changes of the chromatin structure of the HMLalpha locus. The HMLalpha3 gene did not appear to be part of the silent domain because alpha3p was expressed from both MATalpha3 and HMLalpha3 and sir mutations failed to change the chromatin structure of the HMLalpha3 gene. Furthermore, a 102-bp silencer element was isolated from the HMLalpha flanking DNA. HMLalpha was also flanked by an autonomously replicating sequence (ARS) activity, but the ARS activity did not appear to be required for silencer function. K. lactis sir2 strains grown in the presence of ethidium bromide (EtBr) accumulated the drug, which interfered with the essential mitochondrial genome. Mutations that bypassed the requirement for the mitochondrial genome also bypassed the EtBr sensitivity of sir2 strains. Sir2p localized to the nucleus, indicating that the role of Sir2p to hinder EtBr accumulation was an indirect regulatory effect. Sir2p was also required for growth in the presence of high concentrations of Ni(2+) and Cu(2+). PMID:10978277

Astrm, S U; Kegel, A; Sjstrand, J O; Rine, J

2000-01-01

252

Characterization, expression, and mutation of the Lactococcus lactis galPMKTE genes, involved in galactose utilization via the Leloir pathway  

Microsoft Academic Search

A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose 1-epimerase (GalM), a galactoki- nase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GalE), re-

B. P. Groossiord; Evert J. Luesink; Elaine E. Vaughan; Alain Arnaud; Vos de W. M

2003-01-01

253

Time-Resolved Determination of the CcpA Regulon of Lactococcus lactis subsp. cremoris MG1363?  

PubMed Central

Carbon catabolite control protein A (CcpA) is the main regulator involved in carbon catabolite repression in gram-positive bacteria. Time series gene expression analyses of Lactococcus lactis MG1363 and L. lactis MG1363?ccpA using DNA microarrays were used to define the CcpA regulon of L. lactis. Based on a comparison of the transcriptome data with putative CcpA binding motifs (cre sites) in promoter sequences in the genome of L. lactis, 82 direct targets of CcpA were predicted. The main differences in time-dependent expression of CcpA-regulated genes were differences between the exponential and transition growth phases. Large effects were observed for carbon and nitrogen metabolic genes in the exponential growth phase. Effects on nucleotide metabolism genes were observed primarily in the transition phase. Analysis of the positions of putative cre sites revealed that there is a link between either repression or activation and the location of the cre site within the promoter region. Activation was observed when putative cre sites were located upstream of the hexameric ?35 sequence at an average position of ?56.5 or further upstream with decrements of 10.5 bp. Repression was observed when the cre site was located in or downstream of putative ?35 and ?10 sequences. The highest level of repression was observed when the cre site was present at a defined side of the DNA helix relative to the canonical ?10 sequence. Gel retardation experiments, Northern blotting, and enzyme assays showed that CcpA represses its own expression and activates the expression of the divergently oriented prolidase-encoding pepQ gene, which constitutes a link between regulation of carbon metabolism and regulation of nitrogen metabolism. PMID:17028270

Zomer, Aldert L.; Buist, Girbe; Larsen, Rasmus; Kok, Jan; Kuipers, Oscar P.

2007-01-01

254

Characterization of the d-Xylulose 5-Phosphate/d-Fructose 6-Phosphate Phosphoketolase Gene (xfp) from Bifidobacterium lactis  

PubMed Central

A d-xylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase (Xfp) from the probiotic Bifidobacterium lactis was purified to homogeneity. The specific activity of the purified enzyme with d-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme. Km values for d-xylulose 5-phosphate and d-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (named xfp) encoding 825 amino acids. The xfp gene was identified on the chromosome of B. lactis with the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from the xfp gene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes, pta and guaA, were localized adjacent to xfp on the B. lactis chromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes in Mycobacterium tuberculosis. However, xfp is transcribed in B. lactis as a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase. PMID:11292814

Meile, Leo; Rohr, Lukas M.; Geissmann, Thomas A.; Herensperger, Monique; Teuber, Michael

2001-01-01

255

Characterization of the D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase gene (xfp) from Bifidobacterium lactis.  

PubMed

A D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase (Xfp) from the probiotic Bifidobacterium lactis was purified to homogeneity. The specific activity of the purified enzyme with D-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme. K(m) values for D-xylulose 5-phosphate and D-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (named xfp) encoding 825 amino acids. The xfp gene was identified on the chromosome of B. lactis with the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from the xfp gene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes, pta and guaA, were localized adjacent to xfp on the B. lactis chromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes in Mycobacterium tuberculosis. However, xfp is transcribed in B. lactis as a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase. PMID:11292814

Meile, L; Rohr, L M; Geissmann, T A; Herensperger, M; Teuber, M

2001-05-01

256

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis  

Microsoft Academic Search

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either

SANDRA TORRIANI; GIACOMO ZAPPAROLI; FRANCO DELLAGLIO

1999-01-01

257

Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to fermentative metabolism by deletion of NDI1.  

PubMed

In this paper, we report the metabolic engineering of the respiratory yeast Kluyveromyces lactis by construction and characterization of a null mutant (?klndi1) in the single gene encoding a mitochondrial alternative internal dehydrogenase. Isolated mitochondria of the ?klndi1 mutant show unaffected rate of oxidation of exogenous NADH, but no oxidation of matrix NADH; this confirms that KlNdi1p is the only internal NADH dehydrogenase in K.?lactis mitochondria. Permeabilized cells of the ?klndi1 mutant do not show oxidation of matrix NADH, which suggests that shuttle systems to transfer the NADH from mitochondrial matrix to cytosol, for being oxidized by external dehydrogenases, are not functional. The ?klndi1 mutation decreases the chronological life span in absence of nutrients. The expression of KlNDI1 is increased by glutathione reductase depletion. The ?klndi1 mutation shifts the K.?lactis metabolism from respiratory to fermentative: the ?klndi1 strain shows reduced respiration rate and increased ethanol production from glucose, while it does not grow in non-fermentable carbon sources such as lactate. The biotechnological benefit of the ?klndi1 mutant for bioethanol production from waste cheese whey lactose was proved. PMID:25186243

Gonzlez-Siso, Mara Isabel; Tourio, Alba; Vizoso, Angel; Pereira-Rodrguez, Angel; Rodrguez-Belmonte, Esther; Becerra, Manuel; Cerdn, Mara Esperanza

2014-09-01

258

The deletion of the succinate dehydrogenase gene KlSDH1 in Kluyveromyces lactis does not lead to respiratory deficiency.  

PubMed

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose. PMID:15189981

Saliola, Michele; Bartoccioni, Paola Chiara; De Maria, Ilaria; Lodi, Tiziana; Falcone, Claudio

2004-06-01

259

Efficient production of secreted staphylococcal antigens in a non-lysing and proteolytically reduced Lactococcus lactis strain.  

PubMed

Cell surface-exposed and secreted proteins are attractive targets for vaccination against pathogenic gram-positive bacteria. To obtain sufficient amounts of such antigens, efficient protein production platforms are needed. In this study, a pipeline for the production and purification of surface-exposed and secreted antigens of the gram-positive bacterial pathogen Staphylococcus aureus is presented. Cytoplasmic or extracellular production of S. aureus antigens was achieved using the Lactococcus lactis strain PA1001, which lacks the major extracellular protease HtrA and the autolysin AcmA to minimize proteolysis and cell lysis, respectively. For most tested S. aureus antigens, secretory production directed by the signal peptide of the major secreted protein Usp45 of L. lactis resulted in higher yields than intracellular production without a signal peptide. Additionally, secretory production of His-tagged antigens allowed their facile one-step purification from the growth medium by metal affinity chromatography. For three of the purified antigens, biological activity was confirmed through enzyme activity assays. We, furthermore, show that the present pipeline can be used to produce staphylococcal antigens with an N-terminal AVI-tag for site-specific labeling with biotin or a C-terminal cell wall-binding domain for cell surface display. We conclude that our L. lactis-based pipeline allows the efficient production of S. aureus antigens and their subsequent purification in one step. PMID:25176446

Neef, Jolanda; Koedijk, Danny G A M; Bosma, Tjibbe; van Dijl, Jan Maarten; Buist, Girbe

2014-12-01

260

The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency  

PubMed Central

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose. PMID:15189981

Saliola, Michele; Bartoccioni, Paola Chiara; De Maria, Ilaria; Lodi, Tiziana; Falcone, Claudio

2004-01-01

261

Genome-Scale Genotype-Phenotype Matching of Two Lactococcus lactis Isolates from Plants Identifies Mechanisms of Adaptation to the Plant Niche?  

PubMed Central

Lactococcus lactis is a primary constituent of many starter cultures used for the manufacturing of fermented dairy products, but the species also occurs in various nondairy niches such as (fermented) plant material. Three genome sequences of L. lactis dairy strains (IL-1403, SK11, and MG1363) are publicly available. An extensive molecular and phenotypic diversity analysis was now performed on two L. lactis plant isolates. Diagnostic sequencing of their genomes resulted in over 2.5 Mb of sequence for each strain. A high synteny was found with the genome of L. lactis IL-1403, which was used as a template for contig mapping and locating deletions and insertions in the plant L. lactis genomes. Numerous genes were identified that do not have homologs in the published genome sequences of dairy L. lactis strains. Adaptation to growth on substrates derived from plant cell walls is evident from the presence of gene sets for the degradation of complex plant polymers such as xylan, arabinan, glucans, and fructans but also for the uptake and conversion of typical plant cell wall degradation products such as ?-galactosides, ?-glucosides, arabinose, xylose, galacturonate, glucuronate, and gluconate. Further niche-specific differences are found in genes for defense (nisin biosynthesis), stress response (nonribosomal peptide synthesis and various transporters), and exopolysaccharide biosynthesis, as well as the expected differences in various mobile elements such as prophages, plasmids, restriction-modification systems, and insertion sequence elements. Many of these genes were identified for the first time in Lactococcus lactis. In most cases good correspondence was found with the phenotypic characteristics of these two strains. PMID:18039825

Siezen, Roland J.; Starrenburg, Marjo J. C.; Boekhorst, Jos; Renckens, Bernadet; Molenaar, Douwe; van Hylckama Vlieg, Johan E. T.

2008-01-01

262

Efficient Overproduction of Membrane Proteins in Lactococcus lactis Requires the Cell Envelope Stress Sensor/Regulator Couple CesSR  

PubMed Central

Background Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein. Methods and Findings We describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1?9-H6 more than 4-fold. Conclusions The results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins. PMID:21818275

Pinto, Joao P. C.; Kuipers, Oscar P.; Marreddy, Ravi K. R.; Poolman, Bert; Kok, Jan

2011-01-01

263

Bonds between Fibronectin and Fibronectin-Binding Proteins on Staphylococcus aureus and Lactococcus lactis  

PubMed Central

Bacterial cell-wall-associated fibronectin binding proteins A and B (FnBPA and FnBPB) form bonds with host fibronectin. This binding reaction is often the initial step in prosthetic device infections. Atomic force microscopy was used to evaluate binding interactions between a fibronectin-coated probe and laboratory-derived Staphylococcus aureus that are (i) defective in both FnBPA and FnBPB (fnbA fnbB double mutant, DU5883), (ii) capable of expressing only FnBPA (fnbA fnbB double mutant complemented with pFNBA4), or (iii) capable of expressing only FnBPB (fnbA fnbB double mutant complemented with pFNBB4). These experiments were repeated using Lactococcus lactis constructs expressing fnbA and fnbB genes from S. aureus. A distinct force signature was observed for those bacteria that expressed FnBPA or FnBPB. Analysis of this force signature with the biomechanical wormlike chain model suggests that parallel bonds form between fibronectin and FnBPs on a bacterium. The strength and covalence of bonds were evaluated via nonlinear regression of force profiles. Binding events were more frequent (p < 0.01) for S. aureus expressing FnBPA or FnBPB than for the S. aureus double mutant. The binding force, frequency, and profile were similar between the FnBPA and FnBPB expressing strains of S. aureus. The absence of both FnBPs from the surface of S. aureus removed its ability to form a detectable bond with fibronectin. By contrast, ectopic expression of FnBPA or FnBPB on the surface of L. lactis conferred fibronectin binding characteristics similar to those of S. aureus. These measurements demonstrate that fibronectin-binding adhesins FnBPA and FnBPB are necessary and sufficient for the binding of S. aureus to prosthetic devices that are coated with host fibronectin. PMID:20218549

2010-01-01

264

Putrescine production via the agmatine deiminase pathway increases the growth of Lactococcus lactis and causes the alkalinization of the culture medium.  

PubMed

Lactococcus lactis is the most important starter culture organism used in the dairy industry. Although L. lactis species have been awarded Qualified Presumption of Safety status by the European Food Safety Authority, and Generally Regarded as Safe status by the US Food and Drug Administration, some strains can produce the biogenic amine putrescine. One such strain is L. lactis subsp. cremoris CECT 8666 (formerly L. lactis subsp. cremoris GE2-14), which was isolated from Genestoso cheese. This strain catabolizes agmatine to putrescine via the agmatine deiminase (AGDI) pathway, which involves the production of ATP and two ammonium ions. The present work shows that the availability of agmatine and its metabolization to putrescine allows for greater bacterial growth (in a biphasic pattern) and causes the alkalinization of the culture medium in a dose-dependent manner. The construction of a mutant lacking the AGDI cluster (L. lactis CECT 8666 ?agdi) confirmed the latter's direct role in putrescine production, growth, and medium alkalinization. Alkalinization did not affect the putrescine production pattern and was not essential for increased bacterial growth. PMID:25341400

Del Rio, Beatriz; Linares, Daniel M; Ladero, Victor; Redruello, Begoa; Fernndez, Maria; Martin, Maria Cruz; Alvarez, Miguel A

2014-10-24

265

Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity.  

PubMed

Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium. As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate. This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio. The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined. Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio. Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism. PMID:10937934

Even, S; Garrigues, C; Loubiere, P; Lindley, N D; Cocaign-Bousquet, M

1999-07-01

266

KNQ1, a Kluyveromyces lactis gene encoding a transmembrane protein, may be involved in iron homeostasis.  

PubMed

The original purpose of the experiments described in this article was to identify, in the biotechnologically important yeast Kluyveromyces lactis, gene(s) that are potentially involved in oxidative protein folding within the endoplasmic reticulum (ER), which often represents a bottleneck for heterologous protein production. Because treatment with the membrane-permeable reducing agent dithiothreitol inhibits disulfide bond formation and mimics the reducing effect that the normal transit of folding proteins has in the ER environment, the strategy was to search for genes that conferred higher levels of resistance to dithiothreitol when present in multiple copies. We identified a gene (KNQ1) encoding a drug efflux permease for several toxic compounds that in multiple copies conferred increased dithiothreitol resistance. However, the KNQ1 product is not involved in the excretion of dithiothreitol or in recombinant protein secretion. We generated a knq1 null mutant, and showed that both overexpression and deletion of the KNQ1 gene resulted in increased resistance to dithiothreitol. KNQ1 amplification and deletion resulted in enhanced transcription of iron transport genes, suggesting, for the membrane-associated protein Knq1p, a new, unexpected role in iron homeostasis on which dithiothreitol tolerance may depend. PMID:17428309

Marchi, Emmanuela; Lodi, Tiziana; Donnini, Claudia

2007-08-01

267

pH-Rate Profiles Support a General Base Mechanism for Galactokinase (Lactococcus lactis)  

PubMed Central

Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the conversion of ?-D-galactose to galactose-1-phosphate. For this investigation, we studied the kinetic mechanism and pH profiles of the enzyme from Lactococcus lactis. Our results show that the mechanism for its reaction is sequential in both directions. Mutant proteins D183A and D183N are inactive (<10,000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C-1 hydroxyl group of galactose. The pHkcat profile of the forward reaction has a pKa of 6.9 0.2 that likely is due to Asp183. The pH-kcat/KGal profile of the reverse reaction further substantiates this role as it is lacking a key pKa required for a direct proton transfer mechanism. The R36A and R36N mutant proteins show over 100-fold lower activity than that for the wild-type enzyme, thus suggesting that Arg36 lowers the pKa of the C-1 hydroxyl to facilitate deprotonation. PMID:23872454

Reinhardt, Laurie A.; Thoden, James B.; Peters, Greg S.; Holden, Hazel M.; Cleland, W.W.

2013-01-01

268

Survival, Physiology, and Lysis of Lactococcus lactis in the Digestive Tract  

PubMed Central

The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration. For this purpose, we used strains marked with reporter genes, the luxA-luxB gene of Vibrio harveyi and the gfp gene of Aequora victoria, that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria. Luciferase was chosen to measure the metabolic activity of Lactococcus lactis in the digestive tract because it requires NADH, which is available only in metabolically active cells. The green fluorescent protein was used to assess the bacterial lysis independently of death. We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact. Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival). In contrast, only 10 to 30% of bacteria survive in the duodenum. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm. This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies. PMID:10543799

Drouault, Sophie; Corthier, Grard; Ehrlich, S. Dusko; Renault, Pierre

1999-01-01

269

Dual inducible expression of recombinant GFP and targeted antisense RNA in Lactococcus lactis.  

PubMed

The food-grade status and probiotic activity of lactic acid bacteria (LAB) make them attractive hosts for production and oral delivery of therapeutic heterologous vaccines and other proteins, yet these bacteria currently do not achieve recombinant protein expression at levels comparable to those seen in Escherichia coli and Saccharomyces cerevisiae. Limited levels of expressed recombinant protein per cell most likely constrain the vaccine's immunogenic potential with respect to the magnitude and specificity of the immune response. With the goal of increasing recombinant protein expression per cell in Lactococcus lactis IL1403, a model LAB, we have constructed and evaluated a new vector that permits simultaneously-induced expression of GFP, a model recombinant protein, and antisense RNA inhibition of the clpP-encoded intracellular protease. While silencing of the rational target clpP does not lead to increased GFP per cell, the new dual-expression system provides an efficient and potentially high-throughput metabolic engineering tool for strain improvement. PMID:19523486

Oddone, Gian M; Mills, David A; Block, David E

2009-09-01

270

Effect of exopolysaccharide produced by isogenic strains of Lactococcus lactis on half-fat Cheddar cheese.  

PubMed

Fat-reduced cheeses often suffer from undesirable texture, flavor, and cooking properties. Exopolysaccharides (EPS) produced by starter strains have been proposed as a mechanism to increase yield and to improve the texture and cooking properties of reduced-fat cheeses. The objective of this work was to assess the influence of an exopolysaccharide on the yield, texture, cooking properties, and quality of half-fat Cheddar cheese. Two pilot-scale half-fat Cheddar cheeses were manufactured using single starters of an isogenic strain of Lactococcus lactis ssp. cremoris (DPC6532 and DPC6533) that differed in their ability to produce exopolysaccharide. Consequently, any differences detected between the cheeses were attributed to the presence of the exopolysaccharide. The results indicated that cheeses made with the exopolysaccharide-producing starter had an 8.17% increase in actual cheese yield (per 100 kg of milk), a 9.49% increase in moisture content, increase in water activity and water desorption rate at relative humidities

Costa, N E; Hannon, J A; Guinee, T P; Auty, M A E; McSweeney, P L H; Beresford, T P

2010-08-01

271

Oral immunization with recombinant hepatitis E virus antigen displayed on the Lactococcus lactis surface enhances ORF2-specific mucosal and systemic immune responses in mice.  

PubMed

Hepatitis E virus (HEV) as a recognized zoonotic pathogen has posed global burden on public health, which is exacerbated by lack of efficient vaccine. In this study, we constructed a recombinant (inaQ-ORF2 gene fusion) Lactococcus lactis (L. lactis) strain NZ3900 that expresses and displays the hepatitis E virus antigen ORF2 utilizing an ice uncleation protein-based anchor system. After oral vaccination of BALB/c mice, significantly higher levels of ORF2-specific mucosal IgA and serum IgG were detected and cellular immunity was also induced. These findings further support that L. lactis-based HEV antigen vaccines could be used for human and animal protection against infection. PMID:25445956

Gao, Shenyang; Li, Dandan; Liu, Ying; Zha, Enhui; Zhou, Tiezhong; Yue, Xiqing

2015-01-01

272

Cloning and functional expression of the mitochondrial alternative oxidase gene (aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions.  

PubMed

Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions. There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study, a cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for alternative respiration, from a citric acid producing Aspergillus niger strain was cloned and expressed in L. lactis as a host strain. Expression of aox1 conferred on this organism cyanide-resistant and salicylhydroxamate-sensitive growth. Bioreactor cultures under fully aerobic conditions of the transformed L. lactis showed that the alternative respiratory pathway operates and improves significantly the microorganism's response to oxidizing stress conditions as it enhances biomass production, suppresses lactate formation, and leads to accumulation of large amounts of nisin. PMID:22133435

Papagianni, Maria; Avramidis, Nicholaos

2012-01-01

273

Lactococcus lactis carrying the pValac DNA expression vector coding for IL-10 reduces inflammation in a murine model of experimental colitis  

PubMed Central

Background Inflammatory bowel diseases (IBD) are intestinal disorders characterized by inflammation in the gastrointestinal tract. Interleukin-10 is one of the most important anti-inflammatory cytokines involved in the intestinal immune system and because of its role in downregulating inflammatory cascades, its potential for IBD therapy is under study. We previously presented the development of an invasive strain of Lactococcus lactis (L. lactis) producing Fibronectin Binding Protein A (FnBPA) which was capable of delivering, directly to host cells, a eukaryotic DNA expression vector coding for IL-10 of Mus musculus (pValac:il-10) and diminish inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of intestinal inflammation. As a new therapeutic strategy against IBD, the aim of this work was to evaluate the therapeutic effect of two L. lactis strains (the same invasive strain evaluated previously and the wild-type strain) carrying the therapeutic pValac:il-10 plasmid in the prevention of inflammation in a dextran sodium sulphate (DSS)-induced mouse model. Results Results obtained showed that not only delivery of the pValac:il-10 plasmid by the invasive strain L. lactis MG1363 FnBPA+, but also by the wild-type strain L. lactis MG1363, was effective at diminishing intestinal inflammation (lower inflammation scores and higher IL-10 levels in the intestinal tissues, accompanied by decrease of IL-6) in the DSS-induced IBD mouse model. Conclusions Administration of both L. lactis strains carrying the pValac:il-10 plasmid was effective at diminishing inflammation in this murine model of experimental colitis, showing their potential for therapeutic intervention of IBD. PMID:25106058

2014-01-01

274

Cloning, production, and functional expression of the bacteriocin enterocin A, produced by Enterococcus faecium T136, by the yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans.  

PubMed

The bacteriocin enterocin A (EntA) produced by Enterococcus faecium T136 has been successfully cloned and produced by the yeasts Pichia pastoris X-33EA, Kluyveromyces lactis GG799EA, Hansenula polymorpha KL8-1EA, and Arxula adeninivorans G1212EA. Moreover, P. pastoris X-33EA and K. lactis GG799EA produced EntA in larger amounts and with higher antimicrobial and specific antimicrobial activities than the EntA produced by E. faecium T136. PMID:22685156

Borrero, Juan; Kunze, Gotthard; Jimnez, Juan J; Ber, Erik; Gtiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernndez, Pablo E

2012-08-01

275

The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model  

PubMed Central

Background Oral immunization with vaccines may be an effective strategy for prevention of Clostridium difficile infection (CDI). However, application of previously developed vaccines for preventing CDI has been limited due to various reasons. Here, we developed a recombinant Lactococcus lactis oral vaccine and evaluated its effect on a C. difficile-infected animal model established in golden hamsters in attempt to provide an alternative strategy for CDI prevention. Methods Recombinant L. lactis vaccine was developed using the pTRKH2 plasmid, a high-copy-number Escherichia coli-L. shuttle vector: 1) L. lactis expressing secreted proteins was constructed with recombinant pTRKH2 (secreted-protein plasmid) carrying the Usp45 signal peptide (SPUsp45), nontoxic adjuvanted tetanus toxin fragment C (TETC), and 14 of the 38 C-terminal repeats (14CDTA) of nontoxic C. difficile toxin A (TcdA); and 2) L. lactis expressing secreted and membrane proteins was constructed with recombinant pTRKH2 (membrane-anchored plasmid) carrying SPUsp45, TETC, 14CDTA, and the cell wall-anchored sequence of protein M6 (cwaM6). Then, 32 male Syrian golden hamsters were randomly divided into 4 groups (n?=?8 each) for gavage of normal saline (blank control) and L. lactis carrying the empty shuttle vector, secreted-protein plasmid, and membrane-anchored plasmid, respectively. After 1-week gavage of clindamycin, the animals were administered with C. difficile spore suspension. General symptoms and intestinal pathological changes of the animals were examined by naked eye and microscopy, respectively. Protein levels of anti-TcdA IgG/IgA antibodies in intestinal tissue and fluid were analyzed by enzyme-linked immunosorbent assay (ELISA). A cell culture cytotoxicity neutralization assay was done by TcdA treatment with or without anti-TcdA serum pre-incubation or treatment. Apoptosis of intestinal epithelial cells was examined by flow cytometry (FL) assay. Expression of mucosal inflammatory cytokines in the animals was detected by polymer chain reaction (PCR) assay. Results After the C. difficile challenge, the animals of control group had severe diarrhea symptoms on day 1 and all died on day 4, indicating that the CDI animal model was established in hamster. Of the 3 immunization groups, secreted-protein and membrane-anchored plasmid groups had significantly lower mortalities, body weight decreases, and pathological scores, with higher survival rate/time than the empty plasmid group (P??0.05). The anti-TcdA serum of membrane-anchored plasmid group neutralized the cytotoxicity of 200 ng/ml TcdA with the best protective effect achieved by anti-TcdA serum pre-incubation. The incidences of TcdA-induced death and apoptosis of intestinal epithelial cells were significantly reduced by cell pre-incubation or treatment with anti-TcdA serum of membrane-anchored plasmid group (P?lactis live vaccine is effective for preventing CDI in the hamster model, thus providing an alternative for immunization of C. difficile-associated diseases. PMID:23865596

2013-01-01

276

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin.  

PubMed

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively. PMID:16807924

Lan, Christopher Q; Oddone, Gian; Mills, David A; Block, David E

2006-12-20

277

Microencapsulation of Bifidobacterium animalis subsp. lactis in Modified Alginate-chitosan Beads and Evaluation of Survival in Simulated Gastrointestinal Conditions  

Microsoft Academic Search

Bifidobacterium animalis subsp. lactis was entrapped in alginate, alginate-chitosan, alginate-chitosan-Sureteric and alginate-chitosan-Acryl-Eze. Survival and in vitro release of bifidobacteria from the microparticles were investigated under conditions simulating gastrointestinal fluids covering the pH range from 1.5 to 7.5, with and without pepsin (3gL), pancreatin (1gL), and bile (10gL). All types of microcapsules protected B. animalis, but the use of chitosan and

Alcina Maria Liserre; Maria Ines R; Bernadette D. G. M. Franco

2007-01-01

278

A System To Generate Chromosomal Mutations inLactococcus lactisWhich Allows Fast Analysis of Targeted Genes  

Microsoft Academic Search

pWV01 derivative pVE6007. Transformation of L. lactis MG1363(pVE6007) with the pORI19 bank of lacto- coccal chromosomal fragments at the permissive temperature allowed replication of several copies of a recombinant plasmid from the bank within a cell because of the provision intransof RepA-Ts from pVE6007. A temperature shift to 37&C resulted in loss of pVE6007 and integration of the pORI19 derivatives

JEAN LAW; GIRBE BUIST; ALFRED HAANDRIKMAN; JAN KOK; GERARD VENEMA; ANDKEES LEENHOUTS

1995-01-01

279

Inverted terminal repetitions of the two linear DNA associated with the killer character of the yeast Kluyveromyces lactis.  

PubMed Central

The killer character of some Kluyveromyces lactis strains is associated with the presence of two linear double-stranded DNA, pGKl-1 (or k1) and pGKl-2 (or k2). Nucleotide sequencing has revealed that each DNA has inverted terminal repetitions of about 200 base-pairs whose 5' ends seem to be blocked. The repetitions of the two DNA do not share extensive sequence homology. The role of these repetitions in the replication of killer DNA is discussed. Images PMID:6878039

Sor, F; Wsolowski, M; Fukuhara, H

1983-01-01

280

A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis  

PubMed Central

Background The Crabtree-negative yeast species Kluyveromyces lactis has been established as an attractive microbial expression system for recombinant proteins at industrial scale. Its LAC genes allow for utilization of the inexpensive sugar lactose as a sole source of carbon and energy. Lactose efficiently induces the LAC4 promoter, which can be used to drive regulated expression of heterologous genes. So far, strain manipulation of K. lactis by homologous recombination was hampered by the high rate of non-homologous end-joining. Results Selection for growth on lactose was applied to target the insertion of heterologous genes downstream of the LAC4 promoter into the K. lactis genome and found to yield high numbers of positive transformants. Concurrent reconstitution of the ?-galactosidase gene indicated the desired integration event of the expression cassette, and ?-galactosidase activity measurements were used to monitor gene expression for strain improvement and fermentation optimization. The system was particularly improved by usage of a cell lysis resistant strain, VAK367-D4, which allowed for protein accumulation in long-term fermentation. Further optimization was achieved by increased gene dosage of KlGAL4 encoding the activator of lactose and galactose metabolic genes that led to elevated transcription rates. Pilot experiments were performed with strains expressing a single-chain antibody fragment (scFvox) and a viral envelope protein (BVDV-E2), respectively. scFvox was shown to be secreted into the culture medium in an active, epitope-binding form indicating correct processing and protein folding; the E2 protein could be expressed intracellularly. Further data on the influence of protein toxicity on batch fermentation and potential post-transcriptional bottlenecks in protein accumulation were obtained. Conclusions A novel Kluyveromyces lactis host-vector system was developed that places heterologous genes under the control of the chromosomal LAC4 promoter and that allows monitoring of its transcription rates by ?-galactosidase measurement. The procedure is rapid and efficient, and the resulting recombinant strains contain no foreign genes other than the gene of interest. The recombinant strains can be grown non-selectively in rich medium and stably maintained even when the gene product exerts protein toxicity. PMID:22905717

2012-01-01

281

Fermentation-induced variation in heat and oxidative stress phenotypes of Lactococcus lactis MG1363 reveals transcriptome signatures for robustness.  

PubMed

Background Lactococcus lactis is industrially employed to manufacture various fermented dairy products. The most cost-effective method for the preservation of L. lactis starter cultures is spray drying, but during this process cultures encounter heat and oxidative stress, typically resulting in low survival rates. However, viability of starter cultures is essential for their adequate contribution to milk fermentation, supporting the ambition to better understand and improve their robustness phenotypes.ResultsThis study describes a transcriptome-phenotype matching approach in which the starter L. lactis MG1363 was fermented under a variety of conditions that differed in the levels of oxygen and/or salt, as well as the fermentation pH and temperature. Samples derived from these fermentations in the exponential phase of bacterial growth were analyzed by full-genome transcriptomics and the assessment of heat and oxidative stress phenotypes. Variations in the fermentation conditions resulted in up to 1000-fold differences in survival during heat and oxidative stress. More specifically, aeration during fermentation induced protection against heat stress, whereas a relatively high fermentation temperature resulted in enhanced robustness towards oxidative stress. Concomitantly, oxygen levels and fermentation temperature induced differential expression of markedly more genes when compared with the other fermentation parameters. Correlation analysis of robustness phenotypes and gene expression levels revealed transcriptome signatures for oxidative and/or heat stress survival, including the metC-cysK operon involved in methionine and cysteine metabolism. To validate this transcriptome-phenotype association we grew L. lactis MG1363 in the absence of cysteine which led to enhanced robustness towards oxidative stress.ConclusionsOverall, we demonstrated the importance of careful selection of fermentation parameters prior to industrial processing of starter cultures. Furthermore, established stress genes as well as novel genes were associated with robustness towards heat and/or oxidative stress. Assessment of the expression levels of this group of genes could function as an indicator for enhanced selection of fermentation parameters resulting in improved robustness during spray drying. The increased robustness after growth without cysteine appeared to confirm the role of expression of the metC-cysK operon as an indicator of robustness and suggests that sulfur amino acid metabolism plays a pivotal role in oxidative stress survival. PMID:25366036

Dijkstra, Annereinou R; Alkema, Wynand; Starrenburg, Marjo; Hugenholtz, Jeroen; van Hijum, Sacha; Bron, Peter A

2014-11-01

282

Kluyveromyces lactis: A Suitable Yeast Model to Study Cellular Defense Mechanisms against Hypoxia-Induced Oxidative Stress  

PubMed Central

Studies about hypoxia-induced oxidative stress in human health disorders take advantage from the use of unicellular eukaryote models. A widely extended model is the fermentative yeast Saccharomyces cerevisiae. In this paper, we describe an overview of the molecular mechanisms induced by a decrease in oxygen availability and their interrelationship with the oxidative stress response in yeast. We focus on the differential characteristics between S. cerevisiae and the respiratory yeast Kluyveromyces lactis, a complementary emerging model, in reference to multicellular eukaryotes. PMID:22928082

Gonzlez Siso, M. Isabel; Cerdn, M. Esperanza

2012-01-01

283

Production of Concentrated Lactococcus lactis subsp. cremoris Suspensions in Calcium Alginate Beads  

PubMed Central

The effect of simultaneous modification of medium composition and growth conditions on the production of Lactococcus lactis subsp. cremoris biomass in calcium alginate beads was studied by the response surface method. Statistical methods of data analysis for unbalanced experiments are illustrated. The media tested were whey, whey supplemented with yeast extract and/or meat extract, milk, and the commercial medium Gold Complete (Nordica). Fermentations were performed at 23C under pH control (5.6, 6.0, 6.4, or 6.8). In one complete series, 1% CaCO3 was added to the growth media. There were strong interactions between CaCO3 and media, CaCO3 and pH level, and CaCO3, media, and pH level. In media with CaCO3, all first-order interactions between media, pH, and sampling time were significant. The addition of CaCO3 increased cell counts in whey-meat extract medium, but no significant difference was found with the other media. Uncoupling between growth and acidification occurred between 16 and 22 h. Highest counts were obtained on milk and Gold Complete (6 1010/g). In CaCO3-containing media, pH influenced cell counts only in whey and in Gold Complete (pH 5.6 and 6.0 giving the best results); pH also influenced the bead mass obtained at the end of the fermentation. Biomass production in alginate gels is proposed as a method of obtaining concentrated cell suspensions without centrifugation or filtration. PMID:16348644

Morin, Nicole; Bernier-Cardou, Michle; Champagne, Claude P.

1992-01-01

284

In vivo nuclear magnetic resonance studies of glycolytic kinetics in Lactococcus lactis.  

PubMed

The metabolism of glucose by nongrowing cells of L. lactis strain MG5267 was studied under controlled conditions of pH, temperature, and gas atmosphere (anaerobic and aerobic) using a circulating system coupled to nuclear magnetic resonance (NMR) detection that allowed a noninvasive determination of intracellular pools of intermediate metabolites by 13C-NMR with a time resolution of 30 seconds. In addition, intracellular parameters, such as pH, NTP levels, and concentration of inorganic phosphate in the cytoplasm, could be monitored on-line by 31P-NMR with a time resolution of approx. 3 min. The time course for the concentrations of intracellular fructose 1,6-bisphosphate (FBP), 3-phosphoglycerate (3-PGA), and phosphoenolpyruvate (PEP), together with kinetic measurements of substrate consumption and endproducts formation, were used as a basis for the construction of a mechanistic model for glycolysis. In vivo measurements were complemented with determinations of phosphorylated metabolites in perchloric acid extracts. A top-down model was developed by simplifying the metabolism to the resolution allowed by the experimental data collected by in vivo NMR (grouped in seven metabolic steps). This simplified mechanistic model was adjusted to the metabolite concentrations determined by in vivo NMR. The results obtained led to the rationalization of the dynamics of glucose metabolism as being driven largely by ATP surplus. This excess causes accumulation of FBP due to NAD+ limitation, whose regeneration is dependent on downstream pyruvate reduction. The model was capable of predicting qualitative shifts in the metabolism of glucose when changing from anaerobic to aerobic conditions. PMID:10397856

Neves, A R; Ramos, A; Nunes, M C; Kleerebezem, M; Hugenholtz, J; de Vos, W M; Almeida, J; Santos, H

1999-07-20

285

Solution properties of viilian, the exopolysaccharide from Lactococcus lactis subsp. cremoris SBT 0495.  

PubMed

The exopolysaccharide (EPS) "viilian" was isolated from a large-batch fermentation of Lactococcus lactis subsp. cremoris SBT 0495. After applying a newly developed purification procedure, pure viilian with a weight-averaged molar mass of 2.64 x 10(3) kg/mol was obtained in a yield of 0.6 g/L culture broth. The native EPS, as well as lower molar mass fractions obtained by sonication of the native polymer, were studied by capillary viscometry and size-exclusion chromatography (SEC) coupled to multiangle laser light scattering detection (MALLS). From the viscosity data at various ionic strengths, we extracted a Mark-Houwink-Kuhn-Sakurada exponent a = 0.79, and a Smidsrod B value of 0.03. By application of the Hearst, Bohdaneck, and Odijk models for stiff polymer coils, in connection to the experimental viscosity data, we established the characteristic ratio to be C(infinity) = 44 and the intrinsic persistence length q(0) = 11.5 nm. The rms radii of gyration predicted from each of the models were in good agreement with the experimental radii (e.g., (1/2)(w) = 162 nm for native viilian in 0.2M NaNO(3)), as determined by SEC-MALLS. In addition, the Odijk model predicts correct ionic strength-linear charge density dependence of the rms radius of gyration. From the combined viscosity and SEC-MALLS experiments we concluded that, in dilute aqueous solutions, viilian behaves as an intermediately stiff, random coil polyelectrolyte system. PMID:10861375

Higashimura, M; Mulder-Bosman, B W; Reich, R; Iwasaki, T; Robijn, G W

2000-08-01

286

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis  

PubMed Central

Crystal structures of the unique hexokinase KlHxk1 of the yeast Kluyveromyces lactis were determined using eight independent crystal forms. In five crystal forms, a symmetrical ring-shaped homodimer was observed, corresponding to the physiological dimer existing in solution as shown by small-angle x-ray scattering. The dimer has a head-to-tail arrangement such that the small domain of one subunit interacts with the large domain of the other subunit. Dimer formation requires favorable interactions of the 15 N-terminal amino acids that are part of the large domain with amino acids of the small domain of the opposite subunit, respectively. The head-to-tail arrangement involving both domains of the two KlHxk1 subunits is appropriate to explain the reduced activity of the homodimer as compared with the monomeric enzyme and the influence of substrates and products on dimer formation and dissociation. In particular, the structure of the symmetrical KlHxk1 dimer serves to explain why phosphorylation of conserved residue Ser-15 may cause electrostatic repulsions with nearby negatively charged residues of the adjacent subunit, thereby inducing a dissociation of the homologous dimeric hexokinases KlHxk1 and ScHxk2. Two complex structures of KlHxk1 with bound glucose provide a molecular model of substrate binding to the open conformation and the subsequent classical domain closure motion of yeast hexokinases. The entirety of the novel data extends the current concept of glucose signaling in yeast and complements the induced-fit model by integrating the events of N-terminal phosphorylation and dissociation of homodimeric yeast hexokinases. PMID:20943665

Kuettner, E. Bartholomeus; Kettner, Karina; Keim, Antje; Svergun, Dmitri I.; Volke, Daniela; Singer, David; Hoffmann, Ralf; Mller, Eva-Christina; Otto, Albrecht; Kriegel, Thomas M.; Strter, Norbert

2010-01-01

287

Paenibacillus lactis sp. nov., isolated from raw and heat-treated milk.  

PubMed

Endospore-forming bacteria were recovered from individual packages from different processing lines in a dairy plant during a tenacious periodical contamination of their UHT-milk production. Two colony types were seen, one of which was identified as Bacillus sporothermodurans. Analysis of the 16S rRNA gene of the second colony type placed these isolates within the genus Paenibacillus, with Paenibacillus lautus as the closest known relative. Moreover, over 99 % similarity was observed to the 16S rDNA sequence of MB 2035, a strain isolated previously from raw milk during a survey at dairy farms for very heat-resistant spore-forming bacteria. Nine other potentially closely related strains among the dairy farm isolates were found using rep-PCR typing. The taxonomic positions of these 19 isolates were further investigated using 16S rRNA gene sequencing and DNA-DNA hybridizations of representative strains. All 19 isolates shared a high degree of phenotypic similarity and were easily distinguished from closely related members of the genus. Anteiso-C(15 : 0), C(16 : 0) and iso-C(15 : 0) were among the major fatty acids and the genomic DNA G+C content was 51.6-51.7 mol%. Therefore, based on their phenotypic, phylogenetic and genomic distinctiveness, these 19 strains, isolated from both raw and heat-treated milk, are placed in the genus Paenibacillus as Paenibacillus lactis sp. nov. The type strain is MB 1871(T) (=LMG 21940(T)=DSM 15596(T)). PMID:15143040

Scheldeman, Patsy; Goossens, Karen; Rodriguez-Diaz, Marina; Pil, Annelies; Goris, Johan; Herman, Lieve; De Vos, Paul; Logan, Niall A; Heyndrickx, Marc

2004-05-01

288

Intragastric administration with recombinant Lactococcus lactis producing heme oxygenase-1 prevents lipopolysaccharide-induced endotoxemia in rats.  

PubMed

Gut injury is a pivotal initiating event in the dysfunctional inflammatory response that causes postinjury multiple organ failure. Heme oxygenase-1 (HO-1) is an important enzyme that provides cellular protection against oxidative stress in different in vitro and in vivo systems. In this study, we evaluated the protective effects of intragastrically administered live Lactococcus lactis secreting bioactive HO-1 to treat intestinal mucosal injury induced by lipopolysaccharide in rats. Intragastric administration with this recombinant L. lactis strain led to active delivery of HO-1 at the mucosa and significantly decreased morbidity and mortality of lipopolysaccharide -induced endotoxemia as confirmed by blinded macroscopic and microscopic inflammatory scores (Chiu's grade), myeloperoxidase activity, mortality, and tumor necrosis factor-alpha and IL-10 cytokine stimulation. This protective effect could be abolished by an HO-1 inhibitor, the zinc protoporphyrin-IX. Our results suggest that a food-grade bacterium genetically modified to deliver bioactive HO-1 in situ exerts a protective effect against intestinal mucosal injury in rats with endotoxemia via modulation of the immune system. This novel approach may be beneficial for the maintenance of the intestinal barrier and anti-inflammatory response of the lower intestine. PMID:18422629

Pang, Qingfeng; Ji, Yong; Li, Yun; Bermdez-Humarn, Luis G; Hu, Gang; Zeng, Yinming

2008-06-01

289

YtjE from Lactococcus lactis IL1403 Is a C-S Lyase with ?,?-Elimination Activity toward Methionine  

PubMed Central

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of l-cysteine, l-cystine, and l-cystathionine. YtjE was also shown to exhibit ?,?-elimination activity toward l-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from l-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from l-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties. PMID:16820483

Martnez-Cuesta, M. Carmen; Pelez, Carmen; Eagles, John; Gasson, Michael J.; Requena, Teresa; Hanniffy, Sean B.

2006-01-01

290

The riboflavin transporter RibU in Lactococcus lactis: molecular characterization of gene expression and the transport mechanism.  

PubMed

This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport. PMID:16585736

Burgess, Catherine M; Slotboom, Dirk Jan; Geertsma, Eric R; Duurkens, Ria H; Poolman, Bert; van Sinderen, Douwe

2006-04-01

291

Biological characteristics and probiotic effect of Leuconostoc lactis strain isolated from the intestine of black porgy fish  

PubMed Central

A strain of lactic acid bacteria, Leuconostoc lactis, was isolated from the intestinal tract of black porgy, Sparus macrocephalus, and identified by conventional biochemical characteristics and 16S rDNA gene sequence analysis. The isolated strain had the ability of bile tolerance and resistance to low pH, and survived well in the trypsinase and pepsin solution. But the highly concentrated dose of trypsinase and pepsin affect the viability of the isolated strain. The isolate was resistant to several antibiotics, including Cephalothin, Ceftriaxone, Imipenem and Tobramycin. The isolate could auto-aggregate itself and coaggregate with other bacteria in vitro. The autoaggregation percentage increased to 23.29% after 20 h of incubation. The percentage of coaggregation were respectively 31.21%, 29.44%, 10.74%, 16.49%, 24.36%, 24.41% and 20.99% for Vibrio parahaemolyticus, Listeria monocytogenes, Escherichia coli O157, Salmonella typhimurium, Shigella, Staphylococcus aureus and Proteusbacillus vulgaris after 20 h incubation of a mixed suspension. The supernatant of the strain inhibited the growth of several pathogens, such as V.parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Staphylococcus aureus, Escherichia coli O157, Salmonella typhimurium, Bacillus subtilis, Proteusbacillus vulgaris and Shigella. These results indicated that the isolate, Leuconostoc lactis, might be an attractive candidate for perspectival strain for probiotics in marine aquaculture. PMID:24516418

Zhang, Wei; Liu, Mingqi; Dai, Xianjun

2013-01-01

292

Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment.  

PubMed

Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (3(4)) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.910(11)CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS. PMID:21683588

Qiao, Jian-Jun; Zhang, Yan-Fei; Sun, Li-Fan; Liu, Wei-Wei; Zhu, Hong-Ji; Zhang, Zhijun

2011-09-01

293

Ability of Lactococcus lactis To Export Viral Capsid Antigens: a Crucial Step for Development of Live Vaccines  

PubMed Central

Thefood grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci. PMID:14660377

Dieye, Yakhya; Hoekman, Arjan J. W.; Clier, Florence; Juillard, Vincent; Boot, Hein J.; Piard, Jean-Christophe

2003-01-01

294

Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions.  

PubMed

The purpose of this study was to improve the survival of Bifidobacterium animalis subsp. lactis 10140 during freeze-drying process by microencapsulation, using a special pediatric prebiotics mixture (galactooligosaccharides and fructooligosaccharides). Probiotic microorganisms were encapsulated with a coat combination of prebiotics-calcium-alginate prior to freeze-drying. Both encapsulated and free cells were then freeze-dried in their optimized combinations of skim milk and prebiotics. Response surface methodology (RSM) was used to produce a coating combination as well as drying medium with the highest cell viability during freeze-drying. The optimum encapsulation composition was found to be 2.1% Na-alginate, 2.9% prebiotic, and 21.7% glycerol. Maximum survival predicted by the model was 81.2%. No significant (p?>?0.05) difference between the predicted and experimental values verified the adequacy of final reduced models. The protection ability of encapsulation was then examined over 120days of storage at 4 and 25C and exposure to a sequential model of infantile GIT conditions including both gastric conditions (pH3.0 and 4.0, 90min, 37C) and intestinal conditions (pH7.5, 5h, 37C). Significantly improved cell viability showed that microencapsulation of B. lactis 10140 with the prebiotics was successful in producing a stable symbiotic powdery nutraceutical. PMID:22843029

Shamekhi, Fatemeh; Shuhaimi, Mustafa; Ariff, Arbakariya; Manap, Yazid A

2013-03-01

295

Impact of Aeration and Heme-Activated Respiration on Lactococcus lactis Gene Expression: Identification of a Heme-Responsive Operon?  

PubMed Central

Lactococcus lactis is a widely used food bacterium mainly characterized for its fermentation metabolism. However, this species undergoes a metabolic shift to respiration when heme is added to an aerobic medium. Respiration results in markedly improved biomass and survival compared to fermentation. Whole-genome microarrays were used to assess changes in L. lactis expression under aerobic and respiratory conditions compared to static growth, i.e., nonaerated. We observed the following. (i) Stress response genes were affected mainly by aerobic fermentation. This result underscores the differences between aerobic fermentation and respiration environments and confirms that respiration growth alleviates oxidative stress. (ii) Functions essential for respiratory metabolism, e.g., genes encoding cytochrome bd oxidase, menaquinone biosynthesis, and heme uptake, are similarly expressed under the three conditions. This indicates that cells are prepared for respiration once O2 and heme become available. (iii) Expression of only 11 genes distinguishes respiration from both aerobic and static fermentation cultures. Among them, the genes comprising the putative ygfCBA operon are strongly induced by heme regardless of respiration, thus identifying the first heme-responsive operon in lactococci. We give experimental evidence that the ygfCBA genes are involved in heme homeostasis. PMID:18487342

Pedersen, Martin Bastian; Garrigues, Christel; Tuphile, Karine; Brun, Clia; Vido, Karin; Bennedsen, Mads; Mllgaard, Henrik; Gaudu, Philippe; Gruss, Alexandra

2008-01-01

296

ClaR-a novel key regulator of cellobiose and lactose metabolism in Lactococcus lactis IL1403.  

PubMed

In a number of previous studies, our group has discovered an alternative pathway for lactose utilization in Lactococcus lactis that, in addition to a sugar-hydrolyzing enzyme with both P-?-glucosidase and P-?-galactosidase activity (BglS), engages chromosomally encoded components of cellobiose-specific PTS (PTS(Cel-Lac)), including PtcA, PtcB, and CelB. In this report, we show that this system undergoes regulation via ClaR, a novel activator protein from the RpiR family of transcriptional regulators. Although RpiR proteins are widely distributed among lactic acid bacteria, their roles have yet to be confirmed by functional assays. Here, we show that ClaR activity depends on intracellular cellobiose-6-phosphate availability, while other sugars such as glucose or galactose have no influence on it. We also show that ClaR is crucial for activation of the bglS and celB expression in the presence of cellobiose, with some limited effects on ptcA and ptcB activation. Among 190 of carbon sources tested, the deletion of claR reduces L. lactis growth only in lactose- and/or cellobiose-containing media, suggesting a narrow specificity of this regulator within the context of sugar metabolism. PMID:25239037

Aleksandrzak-Piekarczyk, Tamara; Stasiak-R?a?ska, Lidia; Cie?la, Jaros?aw; Bardowski, Jacek

2015-01-01

297

The Rag4 glucose sensor is involved in the hypoxic induction of KlPDC1 gene expression in the yeast Kluyveromyces lactis.  

PubMed

Kluyveromyces lactis is a yeast which cannot grow under strict anaerobiosis. To date, no factors responsible for oxygen sensing and oxygen-dependent regulation of metabolism have been identified. In this paper we present the identification of the glucose sensor Rag4 as a factor essential for oxygen-dependent regulation of the fermentative pathway. PMID:21097667

Micolonghi, C; Wsolowski-Louvel, M; Bianchi, M M

2011-01-01

298

Cloning and functional expression of the mitochondrial alternative oxidase gene ( aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions  

Microsoft Academic Search

Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions. There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study,

Maria Papagianni; Nicholaos Avramidis

299

Genome Sequence Analysis of the Biogenic Amine-Producing Strain Lactococcus lactis subsp. cremoris CECT 8666 (Formerly GE2-14).  

PubMed

We here report a 2,801,031-bp annotated draft assembly for the Lactococcus lactis subsp. cremoris GE2-14 genome. This dairy strain produces the biogenic amine putrescine. This sequence may help identify the mechanisms regulating putrescine biosynthesis and throw light on ways to reduce its presence in fermented foods. PMID:25342694

Ladero, Victor; Del Rio, Beatriz; Linares, Daniel M; Fernandez, Maria; Mayo, Baltasar; Martin, M Cruz; Alvarez, Miguel A

2014-01-01

300

Genome Sequence Analysis of the Biogenic Amine-Producing Strain Lactococcus lactis subsp. cremoris CECT 8666 (Formerly GE2-14)  

PubMed Central

We here report a 2,801,031-bp annotated draft assembly for the Lactococcus lactis subsp. cremoris GE2-14 genome. This dairy strain produces the biogenic amine putrescine. This sequence may help identify the mechanisms regulating putrescine biosynthesis and throw light on ways to reduce its presence in fermented foods. PMID:25342694

del Rio, Beatriz; Linares, Daniel M.; Fernandez, Maria; Mayo, Baltasar; Martin, M. Cruz; Alvarez, Miguel A.

2014-01-01

301

Growth Phase-Dependent Proteomes of the Malaysian Isolated Lactococcus lactis Dairy Strain M4 Using Label-Free Qualitative Shotgun Proteomics Analysis  

PubMed Central

Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MSE) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications. PMID:24982972

Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Abdul Rahim, Raha; Mahadi, Nor Muhammad; Illias, Rosli Md.

2014-01-01

302

Insights into the Ropy Phenotype of the Exopolysaccharide-Producing Strain Bifidobacterium animalis subsp. lactis A1dOxR  

PubMed Central

The proteome of the ropy strain Bifidobacterium animalis subsp. lactis A1dOxR, compared to that of its nonropy isogenic strain, showed an overproduction of a protein involved in rhamnose biosynthesis. Results were confirmed by gene expression analysis, and this fact agreed with the high rhamnose content of the ropy exopolysaccharide. PMID:23584772

Hidalgo-Cantabrana, Claudio; Snchez, Borja; Moine, Deborah; Berger, Bernard; de los Reyes-Gaviln, Clara G.; Gueimonde, Miguel; Margolles, Abelardo

2013-01-01

303

Acoustic Emission Signal of Lactococcus lactis before and after Inhibition with NaN3 and Infection with Bacteriophage c2  

PubMed Central

The detection of acoustic emission (AE) from Lactococcus lactis, ssp lactis is reported in which emission intensities are used to follow and define metabolic activity during growth in nutrient broths. Optical density (OD) data were also acquired during L. lactis growth at 32C and provided insight into the timing of the AE signals relative to the lag, logarithmic, and stationary growth phases of the bacteria. The inclusion of a metabolic inhibitor, NaN3, into the nutrient broth eliminated bacteria metabolic activity according to the OD data, the absence of which was confirmed using AE data acquisition. The OD and AE data were also acquired before and after the addition of Bacteriophage c2 in L. lactis containing nutrient broths during the early or middle logarithmic phase; c2 phage m.o.i. (Multiplicity of infection) was varied to help differentiate whether the detected AE was from bacteria cells during lysis or from the c2 phage during genome injection into the cells. It is proposed that AE measurements using piezoelectric sensors are sensitive enough to detect bacteria at the amount near 104?cfu/mL, to provide real time data on bacteria metabolic activity and to dynamically monitor phage infection of cells. PMID:24349820

Stencel, John M.; Hicks, Clair D.; Payne, Fred; Ozevin, Didem

2013-01-01

304

The effects of combined dietary probiotics Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 on innate immunity and disease resistance in olive flounder (Paralichthys olivaceus).  

PubMed

The effects of a dietary probiotic mixture containing Lactococcus (Lc.) lactis BFE920 isolated from bean sprout and autochthonous Lactobacillus (Lb.) plantarum FGL0001 originally isolated from the hindgut of olive flounder (Paralichthys olivaceus) were investigated for the purpose of improving the probiotic effects of Lc. lactis BFE920 on the olive flounder. The immunostimulatory, disease protective, and weight gain effects of Lc. lactis BFE920 were significantly improved when olive flounder (average weight 37.51.26g) were fed the probiotic mixture (log10 7.0CFUeach/g feed pellet) for 30 days. Flounder fed the mixture showed improved skin mucus lysozyme activity and phagocytic activity of innate immune cells compared to flounder fed a single probiotic agent or a control diet. While the levels of neutrophil activity in flounder fed the single probiotic agent or the mixture were similar, they were significantly higher than levels in a control group. Additionally, probiotic-fed flounder showed significantly increased expressions of IL-6, IL-8, and TNF-? in the intestine compared to the control group. Following a 30-day period of being fed probiotics or a control diet, the olive flounder were challenged with an i.p. injection of Streptococcus iniae (log10 6.0CFU/fish). The groups fed the mixed probiotics, Lc. lactis BFE920, Lb. plantarum FGL0001, and the control diet had survival rates of 55%, 45%, 35%, and 20%, respectively. Flounder fed the probiotic mixture gained 38.12.8% more body weight compared to flounder fed the control diet during the 30-day study period. These data strongly suggest that a mixture of Lc. lactis BFE920 and Lb. plantarum FGL0001 may serve as an immunostimulating feed additive useful for disease protection in the fish farming industry. PMID:25449382

Beck, Bo Ram; Kim, Daniel; Jeon, Jongsu; Lee, Sun-Min; Kim, Hui Kwon; Kim, Oi-Jin; Lee, Jae Il; Suh, Byung Sun; Do, Hyung Ki; Lee, Kwan Hee; Holzapfel, Wilhelm H; Hwang, Jee Youn; Kwon, Mun Gyeong; Song, Seong Kyu

2015-01-01

305

Use of synthetic genes for cloning, production and functional expression of the bacteriocins enterocin A and bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis.  

PubMed

The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZ?A and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni. PMID:24510220

Jimnez, Juan J; Borrero, Juan; Gtiez, Loreto; Arbulu, Sara; Herranz, Carmen; Cintas, Luis M; Hernndez, Pablo E

2014-06-01

306

Engineering the central pathways in Lactococcus lactis: functional expression of the phosphofructokinase (pfk) and alternative oxidase (aox1) genes from Aspergillus niger in Lactococcus lactis facilitates improved carbon conversion rates under oxidizing conditions.  

PubMed

The present work describes a novel central pathway engineering method that has been designed with the aim to increase the carbon conversion rates under oxidizing conditions in L. lactis fermentations. The nisin producer L. lactis ATCC11454 strain has been genetically engineered by cloning a truncated version of the phosphofructokinase gene (pfk13), along with the pkaC, encoding for the catalytic subunit of cAMP-dependent protein kinase, and the alternative oxidase (aox1) genes of A. niger. Functional expression of the above genes resulted in enhanced PFK activity and the introduction of AOX activity and alternative respiration in the presence of a source of heme in the substrate, under fully aerobic growth conditions. The constructed strain is capable of fermenting high concentrations of glucose as was demonstrated in a series of glucostat fed-batch fermentations with glucose levels maintained at 55, 138 and 277 mM. The high maximum specific uptake rate of glucose of 1.8 mMs(-1)gCDW(-1) at 277 mM glucose is characteristic of the improved ability of the microorganism to handle elevated glucose concentrations under conditions otherwise causing severe reduction of PFK activity. The increased carbon flow through glycolysis led to increased protein synthesis that was reflected in increased biomass and nisin levels. The pfk 13-pkaC-aox1-transformant strain's fermentation at 277 mM glucose gave a final biomass concentration of 7.5 g/l and nisin activity of 14,000 IU/ml which is, compared to the parental strain's production levels at its optimal 55 mM glucose, increased by a factor of 2.34 for biomass and 4.37 for nisin. PMID:22759530

Papagianni, Maria; Avramidis, Nicholaos

2012-08-10

307

Short communication: phenotypic and genetic diversity of wild Lactococcus lactis isolated from traditional Pecorino cheeses of Tuscany.  

PubMed

Wild Lactococcus lactis isolates from traditional Pecorino cheeses in 4 regions of Tuscany were isolated and characterized to evaluate the diversity of autochthonous lactococci. Sixty strains of Lactococcus were clustered by the results of carbohydrate utilization and diagnostic enzyme activity. Twenty-one unique strains were then chosen for characterization of salt and temperature tolerance, as well as acidification and proteolytic activity in milk. Genetic analysis of these strains was performed via 16S ribosomal DNA sequencing and multilocus sequence typing (MLST) to elucidate diversity relative to their location of origin. Phylogenetic analysis showed distinct clustering by region within organism subspecies, and phenotypic properties demonstrated concomitant trends. Multilocus sequence typing thus allowed for the regional distinction of isolates separate from those of previous works, supporting the concept that distinctive regional qualities of cheeses are strongly influenced by microbial ecology. PMID:23548303

Turchi, B; Van Tassell, M L; Lee, A; Nuvoloni, R; Cerri, D; Miller, M J

2013-06-01

308

CTAB-mediated, single-step preparation of competent Escherichia coli, Bifidobacterium sp. and Kluyveromyces lactis cells  

PubMed Central

An efficient and reproducible method for transformation depends on the competency of the organism. We have developed a simple method for the preparation of competent Escherichia coli, Kluyveromyces lactis, and Bifidobacterium sp. by using a buffer containing cetyl trimethyl ammonium bromide (CTAB) and permits efficient uptake of plasmid DNA and ligation-reaction products. Cells are harvested, washed, mixed with 110?g/ml CTAB, incubated, and followed by a buffer wash. For long-term storage of competent cells, bacteria may be frozen in 10% glycerol without the addition of other components. The transformation process is very simple; plasmid DNA and the cells are mixed and incubated for 560min at 4C; no heat pulse is required, and the duration of incubation at 4C is not crucial. PMID:25606463

Rajagopal, Kammara; Singh, Praveen Kumar; Kumar, Rajesh; Siddiqui, Kaneez Fatima

2014-01-01

309

CTAB-mediated, single-step preparation of competent Escherichia coli, Bifidobacterium sp. and Kluyveromyces lactis cells.  

PubMed

An efficient and reproducible method for transformation depends on the competency of the organism. We have developed a simple method for the preparation of competent Escherichia coli, Kluyveromyces lactis, and Bifidobacterium sp. by using a buffer containing cetyl trimethyl ammonium bromide (CTAB) and permits efficient uptake of plasmid DNA and ligation-reaction products. Cells are harvested, washed, mixed with 1-10?g/ml CTAB, incubated, and followed by a buffer wash. For long-term storage of competent cells, bacteria may be frozen in 10% glycerol without the addition of other components. The transformation process is very simple; plasmid DNA and the cells are mixed and incubated for 5-60min at 4C; no heat pulse is required, and the duration of incubation at 4C is not crucial. PMID:25606463

Rajagopal, Kammara; Singh, Praveen Kumar; Kumar, Rajesh; Siddiqui, Kaneez Fatima

2014-12-01

310

Recombinant Lactococcus lactis can make the difference in antigen-specific immune tolerance induction, the Type 1 Diabetes case  

PubMed Central

Especially in western civilizations, immune diseases that are driven by innocuous (auto- or allo-) antigens are gradually evolving to become pandemic threats. A particularly poignant example is type 1 diabetes, where young children are confronted with the perspective and consequences of total pancreatic ?-cell destruction. Along these disquieting observations we find ourselves equipped with impressively accumulating molecular immunological knowledge on the ins and outs of these pathologies. Often, however, it is difficult to translate this wealth into efficacious medicines. The molecular understanding, the concept of oral tolerance induction, the benefit of using recombinant Lactococcus lactis therein and recent openings towards their clinical use may well enable turning all colors to their appropriate fields on this Rubik's cube. PMID:25185797

2014-01-01

311

Investigation of the Relationship between Lysogeny and Lysis of Lactococcus lactis in Cheese Using Prophage-Targeted PCR  

PubMed Central

The ability of lactococcal strains to lyse (and release intracellular enzymes) during cheese manufacture can be a very desirable trait and has been associated with improvement in flavor and acceleration of cheese ripening. Using a laboratory-scale cheese manufacturing assay, the autolytic behavior of 31 strains of Lactococcus lactis was assessed. In general, marked variation was observed between strains with a 20-fold difference between the best and worst lysing strains based on the release of the intracellular enzyme lactate dehydrogenase. In a parallel experiment, the genomes of these strains were examined for the presence of prophage integrase (int) sequences by using conserved primer sequences from known lysogenic phage. Results demonstrated that the lytic behavior of lactococcal starter strains significantly correlates with the presence of prophage sequences. These results highlight not only the contribution of prophage to starter cell lysis but also the potential of PCR as a useful initial screen to assess strains for this important industrial trait. PMID:10788399

O'Sullivan, David; Ross, R. Paul; Fitzgerald, Gerald F.; Coffey, Aidan

2000-01-01

312

A catalytic and non-catalytic role for the Yen1 nuclease in maintaining genome integrity in Kluyveromyces lactis.  

PubMed

Yen1 is a nuclease identified in Saccharomyces cerevisiae that cleaves the Holliday junction (HJ) intermediate formed during homologous recombination. Alternative routes to disjoin HJs are performed by the Mus81/Mms4- and Sgs1/Top3/Rmi1-complexes. Here, we investigate the role of the Yen1 protein in the yeast Kluyveromyces lactis. We demonstrate that both yen1 mus81 and yen1 sgs1 double mutants displayed negative genetic interactions in the presence of DNA-damaging chemicals. To test if these phenotypes required the catalytic activity of Yen1, we introduced point mutations targeting the catalytic site of Yen1, which abolished the nuclease activity in vitro. Remarkably, catalytically inactive Yen1 did not exacerbate the hydroxyurea sensitivity of the sgs1? strain, which the yen1? allele did. In addition, overexpression of catalytically inactive Yen1 partially rescued the DNA damage sensitivity of both mus81 and sgs1 mutant strains albeit less efficiently than WT Yen1. These results suggest that Yen1 serves both a catalytic and non-catalytic role in its redundant function with Mus81 and Sgs1. Diploids lacking Mus81 had a severe defect in sporulation efficiency and crossover frequency, but diploids lacking both Mus81 and Yen1 showed no further reduction in spore formation. Hence, Yen1 had no evident role in meiosis. However, overexpression of WT Yen1, but not catalytically inactive Yen1 partially rescued the crossover defect in mus81/mus81 mutant diploids. Yen1 is a member of the RAD2/XPG-family of nucleases, but genetic analyses revealed no genetic interaction between yen1 and other family members (rad2, exo1 and rad27). In addition, yen1 mutants had normal nonhomologous end-joining efficiency. We discuss the similarities and differences between K. lactis Yen1 and Yen1/GEN1 from other organisms. PMID:22917548

Chen, Jiang; Astrm, Stefan U

2012-10-01

313

Improved Acid Stress Survival of Lactococcus lactis Expressing the Histidine Decarboxylation Pathway of Streptococcus thermophilus CHCC1524*  

PubMed Central

Degradative amino acid decarboxylation pathways in bacteria generate secondary metabolic energy and provide resistance against acid stress. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 was functionally expressed in the heterologous host Lactococcus lactis NZ9000, and the benefits of the newly acquired pathway for the host were analyzed. During growth in M17 medium in the pH range of 56.5, a small positive effect was observed on the biomass yield in batch culture, whereas no growth rate enhancement was evident. In contrast, a strong benefit for the engineered L. lactis strain was observed in acid stress survival. In the presence of histidine, the pathway enabled cells to survive at pH values as low as 3 for at least 2 h, conditions under which the host cells were rapidly dying. The flux through the histidine decarboxylation pathway in cells grown at physiological pH was under strict control of the electrochemical proton gradient (pmf) across the membrane. Ionophores that dissipated the membrane potential (??) and/or the pH gradient (?pH) strongly increased the flux, whereas the presence of glucose almost completely inhibited the flux. Control of the pmf over the flux was exerted by both ?? and ?pH and was distributed over the transporter HdcP and the decarboxylase HdcA. The control allowed for a synergistic effect between the histidine decarboxylation and glycolytic pathways in acid stress survival. In a narrow pH range around 2.5 the synergism resulted in a 10-fold higher survival rate. PMID:22351775

Trip, Hein; Mulder, Niels L.; Lolkema, Juke S.

2012-01-01

314

Immunomodulatory Activity of Lactococcus lactis A17 from Taiwan Fermented Cabbage in OVA-Sensitized BALB/c Mice  

PubMed Central

From fermented Taiwan foods, we have isolated numerous lactic acid bacteria (LAB) of plant origin and investigated their biological activities. This study aimed to investigate the immunomodulatory effect and mechanism of Lactococcus lactis A17 (A17), isolated from Taiwan fermented cabbage, on ovalbumin (OVA)-sensitized mice. Human peripheral blood mononuclear cells were used to verify immune responses of A17 by IFN-? production. Live (A17-A) and heat-killed A17 (A17-H) were orally administered to OVA-sensitized BALB/c mice to investigate their effects on immunoglobulin (Ig) and cytokine production. The mRNA expression of Toll-like receptors (TLR) and nucleotide binding oligomerization domain (NOD)-like protein receptors in spleen cells was analyzed by real-time RT-PCR. Both live and heat-killed A17 modulate OVA-induced allergic effects. B-cell response was modulated by diminishing IgE production and raising OVA-specific IgG2a production, while T-cell response was modulated by increasing IFN-? production and decreasing IL-4 production. The mRNA expression of NOD-1, NOD-2, and TLR-4 was down-regulated by A17 as well. This is the first report to describe a nave Lactococcus lactis A17 strain as a promising candidate for prophylactic and therapeutic treatments of allergic diseases via oral administration. Our results suggest the ameliorative effects of A17 may be caused by modulating NOD-1 NOD-2, and TLR-4 expression. PMID:23401710

Mei, Hui-Ching; Liu, Yen-Wenn; Chiang, Yi-Chin; Chao, Shiou-Huei; Mei, Nai-Wen; Liu, Yu-Wen; Tsai, Ying-Chieh

2013-01-01

315

Characterisation of the Poly(Vinylpyrrolidone)Poly(Vinylacetate-Co-Crotonic Acid) (PVP:PVAc-CA) Interpolymer Complex Matrix Microparticles Encapsulating a Bifidobacterium lactis Bb12 Probiotic Strain  

Microsoft Academic Search

The method of producing poly-(vinylpyrrolidone)-poly-(vinylacetate-co-crotonic acid) (PVP:PVAc-CA) interpolymer complex matrix\\u000a microparticles in supercritical carbon dioxide (scCO2), encapsulating bacteria, has recently been developed. This study was aimed at probing the external and internal structure\\u000a of these microparticles, which can be used in food. The encapsulation efficiency and distribution of encapsulated Bifidobacterium lactis Bb12 within these microparticles were also investigated. Scanning electron

C. I. Mamvura; F. S. Moolman; L. Kalombo; A. N. Hall; M. S. Thantsha

2011-01-01

316

Binding Properties of Streptococcus gordonii SspA and SspB (Antigen I/II Family) Polypeptides Expressed on the Cell Surface of Lactococcus lactis MG1363  

PubMed Central

The oral bacterium Streptococcus gordonii expresses two cell wall-associated polypeptides, designated SspA (1,542 amino acid residues) and SspB (1,462 amino acid residues), that have 70% sequence identity. These polypeptides are members of the antigen I/II family of oral streptococcal adhesins and mediate the binding of streptococci to salivary glycoproteins, collagen, and other oral microorganisms such as Actinomyces naeslundii. To determine if SspA and SspB have differential binding properties, the coding sequences of the sspA and sspB genes were cloned into expression plasmid vector pTREX1-usp45LS to generate pTREX1-sspA and pTREX1-sspB, respectively, and the Ssp polypeptides were displayed on the cell surface of Lactococcus lactis MG1363. Lactococcal cells expressing similar levels of surface SspA or SspB polypeptide were then compared for their abilities to adhere to a range of antigen I/II polypeptide substrates. More than twice as many L. lactis cells expressing SspA bound to immobilized salivary agglutinin glycoprotein (SAG) as did L. lactis cells expressing SspB. In contrast, lactococci expressing SspB adhered twice as well as lactococci producing SspA to collagen type I and to Candida albicans. The binding of A. naeslundii to lactococci was only weakly enhanced by surface expression of Ssp polypeptides. L. lactis(pTREX1-sspB) cells bound in greater numbers to SAG than did Enterococcus faecalis JH2-2 cells expressing SspB from pAM401EB-5. The results suggest that SspA and SspB have markedly different binding affinities for their oral substrates and thus may function to promote site diversity in colonization by S. gordonii. PMID:9746559

Holmes, Ann R.; Gilbert, Christophe; Wells, Jeremy M.; Jenkinson, Howard F.

1998-01-01

317

Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972  

PubMed Central

Bacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded by Staphylococcus aureus phage vB_SauS-phi-IPLA88, was expressed and secreted in Lactococcus lactis using the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts. PMID:22344638

Rodrguez-Rubio, Lorena; Gutirrez, Dolores; Martnez, Beatriz; Rodrguez, Ana

2012-01-01

318

Lactococcus lactis as a cell factory: a twofold increase in phosphofructokinase activity results in a proportional increase in specific rates of glucose uptake and lactate formation.  

PubMed

Despite the fact that the area of glycolysis in Lactococcus lactis has been intensively studied, only a limited number of studies have been focused on the regulation of uptake of glucose itself. Using the tool of the glucostat fed-batch mode of culture, it was demonstrated in our earlier work that the concentration of glucose regulates its uptake rate and that the control of the glycolytic flux resides to a large extent in processes outside the pathway itself, like glucose transport and the ATP consuming reactions, while allosteric properties of key enzymes like phosphofructokinase (PFK) have a significant influence on the control. Extending our work, we report here the results of fermentations with engineered L. lactis strains with altered PFK activity in which the pfkA gene from Aspergillus niger, and its truncated version pfk13 that encodes a shorter PFK1 fragment were cloned. The results in this study suggest that, under the optimum for the microorganism applied microaerobic conditions, the glycolytic capacity of L. lactis was significantly increased in engineered strains with increased PFK activity. The transformant strain in which the truncated pfk13 gene of A. niger was expressed performed more efficiently as it was able to grow successfully in glucostat cultures with 277 mM glucose - while the optimum glucose concentration for the parental strain was 55 mM. The present work demonstrates the direct effect of PFK activity on the glycolytic flux in L. lactis since a twofold increase in specific PFK activity (from 7.1 to 14.5 U/OD(600)) resulted in a proportional increase of the maximum specific rates of glucose uptake (from 0.8 to 1.7 ?Ms(-1) g CDW(-1)) and lactate formation (from 15 to 22.8 g lactate (g CDW)(-1) h(-1)). PMID:22112409

Papagianni, Maria; Avramidis, Nicholaos

2011-07-10

319

Impact of Bifidobacterium animalis subsp. lactis BB12 and , Lactobacillus acidophilus LA5-containing yoghurt, on fecal bacterial counts of healthy adults  

Microsoft Academic Search

This randomized, placebo-controlled, double blind, parallel doseresponse study investigated the impact of 4-week commercial yoghurt consumption supplemented with Bifidobacterium animalis subsp. lactis (BB-12) and Lactobacillus acidophilus (LA-5) on fecal bacterial counts of healthy adults. Fifty-eight volunteers were randomly assigned to three different groups: 1. placebo (no probiotic, no starter and no green tea extract); 2. Yoptimal (109cfu\\/100g of BB-12 and

Patricia Savard; Benot Lamarche; Marie-Eve Paradis; Hlne Thiboutot; milie Laurin; Denis Roy

2011-01-01

320

Gene cloning, functional expression and secretion of the Slayer protein SgsE from Geobacillus stearothermophilus NRS 2004\\/3a in Lactococcus lactis  

Microsoft Academic Search

The ?93-kDa surface layer protein SgsE of Geobacillus stearothermophilus NRS 2004\\/3a forms a regular crystalline array providing a nanopatterned matrix for the future display of biologically relevant molecules. Lactococcus lactis NZ9000 was established as a safe expression host for the controlled targeted production of SgsE based on the broad host-range plasmid pNZ124Sph, into which the nisA promoter was introduced. SgsE

Andrea Scheberl; Marc Giry-Laterriere; Paul Messner; Christina Schffer

2005-01-01

321

Potential probiotic lactic acid bacteria Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019) do not degrade gastric mucin in vitro  

Microsoft Academic Search

The mucus layer (mucin) coating the surface of the gastrointestinal tract (GIT) plays an important role in the mucosal barrier system. Any damage or disturbance of this mucin layer will compromise the hosts mucosal defence function. In the present study, the ability of three potential probiotic lactic acid bacteria (LAB) strains (Lactobacillus rhamnosus HN001, Lactobacillus acidophilus HN017, Bifidobacterium lactis HN019)

J. S. Zhou; P. K. Gopal; H. S. Gill

2001-01-01

322

Safety assessment of potential probiotic lactic acid bacterial strains Lactobacillus rhamnosus HN001, Lb. acidophilus HN017, and Bifidobacterium lactis HN019 in BALB\\/c mice  

Microsoft Academic Search

The general safety of immune-enhancing lactic acid bacteria (LAB) strains Lactobacillus rhamnosus HN001 (DR20), Lb. acidophilus HN017, and Bifidobacterium lactis HN019 (DR10) was investigated in a feeding trial. Groups of BALB\\/c mice were orally administered test LAB strains or the commercial reference strain Lb. acidophilus LA-1 at 2.5109, 51010 or 2.51012 colony forming units (CFU)\\/kg body weight\\/day for 4 weeks.

J. S Zhou; Q Shu; K. J Rutherfurd; J Prasad; M. J Birtles; P. K Gopal; H. S Gill

2000-01-01

323

In vitro adherence properties of Lactobacillus rhamnosus DR20 and Bifidobacterium lactis DR10 strains and their antagonistic activity against an enterotoxigenic Escherichia coli  

Microsoft Academic Search

Adhesion and colonisation properties of three probiotic strains namely, Lactobacillus rhamnosus DR20, L. acidophilus HN017, and Bifidobacterium lactis DR10, were determined in vitro using the differentiated human intestinal cell-lines including HT-29, Caco-2, and HT29-MTX, and compared with properties of L. acidophilus LA-1 and L. rhamnosus GG (two commercial probiotic strains). Two independent methods were employed to quantitate the adhesiveness of

Pramod K Gopal; Jaya Prasad; John Smart; Harsharanjit S Gill

2001-01-01

324

Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin.  

PubMed

Typical plant aspartic protease zymogens comprise a characteristic and plant-specific insert (PSI). PSI domains can interact with membranes, and a role as a defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet due to problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI, as well as its nonglycosylated mutant. A purification protocol with protein yields of ?4.0 mg/liter was established for both wild-type and nonglycosylated PSIs, which represents the highest reported yield for a nontagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its antifungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins. PMID:24123748

Curto, Pedro; Lufrano, Daniela; Pinto, Ctia; Custdio, Valria; Gomes, Ana Catarina; Trejo, Sebastin A; Baks, Laura; Vairo-Cavalli, Sandra; Faro, Carlos; Simes, Isaura

2014-01-01

325

Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin  

PubMed Central

Typical plant aspartic protease zymogens comprise a characteristic and plant-specific insert (PSI). PSI domains can interact with membranes, and a role as a defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet due to problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI, as well as its nonglycosylated mutant. A purification protocol with protein yields of ?4.0 mg/liter was established for both wild-type and nonglycosylated PSIs, which represents the highest reported yield for a nontagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its antifungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins. PMID:24123748

Curto, Pedro; Lufrano, Daniela; Pinto, Ctia; Custdio, Valria; Gomes, Ana Catarina; Trejo, Sebastin A.; Baks, Laura; Vairo-Cavalli, Sandra; Faro, Carlos

2014-01-01

326

Effect of yogurt containing polydextrose, Lactobacillus acidophilus NCFM and Bifidobacterium lactis HN019: a randomized, double-blind, controlled study in chronic constipation  

PubMed Central

Background Constipation is a frequent complaint and the combination of a prebiotic and probiotics could have a potentially synergic effect on the intestinal transit. The present study therefore aims to investigate the combination of polydextrose (Litesse), L. acidophilus NCFM and B. lactis HN019 in a yogurt on intestinal transit in subjects who suffer from constipation. Methods Patients with constipation were randomly divided into two groups, Control Group (CG) and Treatment Group (TG), and had to eat 180ml of unflavored yogurt every morning for 14days. Those in the CG received only yogurt, while the TG received yogurt containing polydextrose, L. acidophilus NCFM (ATCC 700396) and B. lactis HN019 (AGAL NM97/09513). Results Favourable clinical response was assessed since Agachan score had a significant reduction at the end of the study in both groups and tended to be better in the TG. The subjects in the treatment group also had a shorter transit time at the end of the intervention compared to the control group (p?=?0.01). Conclusion The product containing yogurt with polydextrose, B. lactis HN019 and L. acidophilus NCFM significantly shortened colonic transit time after two weeks in the TG compared to CG and may be an option for treatment of constipation. PMID:25056655

2014-01-01

327

Evaluation of Oral Immunization with Recombinant Avian Influenza Virus HA1 Displayed on the Lactococcus lactis Surface and Combined with the Mucosal Adjuvant Cholera Toxin Subunit B ?  

PubMed Central

The development of safe and efficient avian influenza vaccines for human and animal uses is essential for preventing virulent outbreaks and pandemics worldwide. In this study, we constructed a recombinant (pgsA-HA1 gene fusion) Lactococcus lactis strain that expresses and displays the avian influenza virus HA1 antigens on its surface. The vectors were administered by oral delivery with or without the addition of cholera toxin subunit B (CTB). The resulting immune responses were analyzed, and the mice were eventually challenged with lethal doses of H5N1 viruses. Significant titers of hemagglutinin (HA)-specific serum IgG and fecal IgA were detected in the group that also received CTB. Cellular immunities were also shown in both cell proliferation and gamma interferon (IFN-?) enzyme-linked immunospot (ELISpot) assays. Most importantly, the mice that received the L. lactis pgsA-HA1 strain combined with CTB were completely protected from lethal challenge of the H5N1 virus. These findings support the further development of L. lactis-based avian influenza virus vaccines for human and animal uses. PMID:21632890

Lei, Han; Sheng, Zhina; Ding, Qian; Chen, Jian; Wei, Xiaohui; Lam, Dominic Man-Kit; Xu, Yuhong

2011-01-01

328

Lactococcus lactis BFE920 activates the innate immune system of olive flounder (Paralichthys olivaceus), resulting in protection against Streptococcus iniae infection and enhancing feed efficiency and weight gain in large-scale field studies.  

PubMed

The protective effect of a food-grade lactic acid bacterium Lactococcus lactis BFE920 against disease of olive flounder (Paralichthys olivaceus) cultivated on a large scale was studied. Initially, antimicrobial activity of L. lactis against several fish pathogens was evaluated in vitro; the probiotic showed strong antibacterial activity against Streptococcus iniae, Streptococcus parauberis and Enterococcus viikkiensis, and moderate activity against Lactococcus garviae. When olive flounders were fed for two weeks with experimental diets containing varying concentrations of L. lactis (1 10(6), 5 10(6), 2.5 10(7) and 1.25 10(8) CFU/g feed), all the experimental feed groups showed 68-77% survival upon challenge with S. iniae. A field-scale feeding trial with L. lactis dietary supplement was conducted in a local fish farm (n = 12,000) for three months, and disease resistance, innate immune parameters and growth performance were evaluated. The average weight gain and feed efficiency were increased up to 6.8% and 8.5%, respectively. At the end of the feeding trial, the olive flounders were challenged with S. iniae. The L. lactis-fed group was protected from S. iniae challenge with a 66% survival rate. This disease protection is due to the flounder's innate immunity activated by the L. lactis administration: increased lysosomal activities and production of IL-12 and IFN-?. These data clearly indicated that L. lactis BFE920 may be developed as a functional feed additive for protection against diseases, and for enhancement of feed efficiency and weight gain in olive flounder farming. PMID:24041843

Kim, Daniel; Beck, Bo Ram; Heo, Saet-Byeol; Kim, Jungjoon; Kim, Hyun Duk; Lee, Sun-Min; Kim, Youngchan; Oh, So Young; Lee, Kyungro; Do, HyungKi; Lee, KwanHee; Holzapfel, Wilhelm H; Song, Seong Kyu

2013-11-01

329

Characterization of ?-galacto-oligosaccharides formed via heterologous expression of ?-galactosidases from Lactobacillus reuteri in Lactococcus lactis.  

PubMed

?-Galacto-oligosaccharides (?-GOS) are produced by transgalactosylation reactions of ?-galactosidase (?-Gal) or by conversion of raffinose family oligosaccharides by levansucrase. Similarly to ?-GOS, ?-GOS have the potential to mimic glycan receptors on eukaryotic cells and act as molecular decoys to prevent bacterial infection; however, data on transgalactosylation reactions of ?-Gal remain scarce. The ?-Gal gene sequence from Lactobacillus reuteri was cloned into an ?-Gal negative strain of Lactococcus lactis. Transgalactosylation reactions were achieved using crude cell extracts with melibiose or raffinose as galactosyl donor and fucose, N-acetylglucosamine or lactose as galactosyl acceptor. The composition, sequence and most linkage types of ?-GOS formed with acceptors saccharides were determined by liquid chromatography-tandem mass spectrometry. ?-Gal of Lactobacillus reuteri formed (1???3)-, (1???4)- or (1???6)-linked ?-GOS but exhibited a preference for formation of (1???6)-linkages. Fucose, N-acetylglucosamine and lactose were suitable galactosyl acceptors for ?-Gal of L. reuteri, resulting in formation of (1???3)-, (1???4)- or (1???6)-linked hetero-oligosaccharides. By determining the structural specificity of ?-Gal and increasing the variation of oligosaccharides produced by introducing alternative acceptor sugars, this work supports further studies to assess ?-GOS pathogen adhesion prevention in mammalian hosts. PMID:23942880

Wang, Yvonne; Black, Brenna A; Curtis, Jonathan M; Gnzle, Michael G

2014-03-01

330

A QM/MM Free Energy Study of the Oxidation Mechanism of Dihydroorotate Dehydrogenase (Class 1A) from Lactococcus lactis.  

PubMed

The dihydroorotate dehydrogenase (DHOD) class 1A enzyme catalyzes is the only redox enzyme in the biosynthetic pathway (de novo) of pyrimidines where dihydroorotate (DHO) is oxidized to orotate (OA) coupled to reduction of a flavin mononucleotide (FMN) cofactor. The rupture of two DHO C-H bonds can proceed in a concerted or stepwise way. Herein, the catalytic mechanism of DHOD from Lactococcus lactis involving DHO oxidation (first half-reaction) was described using a hybrid quantum mechanics/molecular mechanics (QM/MM) approach and molecular dynamics simulations. The free energy profile obtained from self-consistent charge-density functional tight binding/molecular mechanics calculations (corrected by DFT/MM) reveals that this occurs with the proton abstraction from DHO C5 to Cys130 deprotonated and DHO H6 is transferred to FMN N5 in a concerted mechanism with a very low barrier of 5.64 kcal/mol. Finally, through a residual decomposition analysis, the residues that have the main influence on the redox reaction were identified. PMID:25564307

Silva, Jos Rogrio A; Roitberg, Adrian E; Alves, Cludio Nahum

2015-01-29

331

Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis.  

PubMed

Microbial production of hyaluronic acid (HA) is an attractive substitute for extraction of this biopolymer from animal tissues. Natural producers such as Streptococcus zooepidemicus are potential pathogens; therefore, production of HA by recombinant bacteria that are generally recognized as safe (GRAS) organisms is a viable alternative that is being extensively explored. However, plasmid-based expression systems for HA production by recombinant bacteria have the inherent disadvantage of reduced productivity because of plasmid instability. To overcome this problem, the HA synthesis genes (hasA-hasB and hasA-hasB-hasC) from has-operon of S. zooepidemicus were integrated into the chromosome of Lactococcus lactis by site-directed, double-homologous recombination developing strains VRJ2AB and VRJ3ABC. The chromosomal integration stabilized the genes and obviated the instability observed in plasmid-expressed recombinant strains. The genome-integrated strains produced higher molecular weight (3.5-4 million Dalton [MDa]) HA compared to the plasmid-expressed strains (2 MDa). High molecular weight HA was produced when the intracellular concentration of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-glucuronic acid (UDP-GlcUA) was almost equal and hasA to hasB ratio was low. This work suggests an optimal approach to obtain high molecular weight HA in recombinant strains. PMID:25044639

Hmar, Rothangmawi Victoria; Prasad, Shashi Bala; Jayaraman, Guhan; Ramachandran, Kadathur B

2014-12-01

332

Stoichiometry of proton movements coupled to ATP synthesis driven by a pH gradient in Streptococcus lactis  

SciTech Connect

An electrochemical potential difference for H+ was established across the plasma membrane of the anaerobe Streptococcus lactis by addition of sulfuric acid to cells suspended in potassium phosphate at pH 8 along with valinomycin or permeant anions. Subsequent acidification of the cell was measured by the distribution of salicyclic acid. A comparison between cells treated or untreated with the inhibitor N,N'-dicyclohexylcarbodiimide was used to reveal that portion of net proton entry attributable to a direct coupling between H+ inflow and synthesis of ATP catalyzed by the reversible proton-translocating ATPase of this microorganism. When the imposed electrochemical proton gradient was below 180-190 mV, proton entry was at the rate expected of passive flux, for both control cells and cells treated with the ATPase inhibitor, However, at higher driving force acidification of control cells was markedly accelerated, coincident with ATP synthesis, while acidification of cells treated with the inhibitor continued at the rate characteristic of passive inflow. This observed threshold (180-190 mV) was identified as the reversal potential for this H+ pump. Parallel measurements showed that the free energy of hydrolysis for ATP in these washed cells was 8.4 kcal/mole (370mV). The comparison between the reversal (threshold) potential and the free energy of hydrolysis for ATP indicates a stoichiometry of 2 H+/ATP for the coupling of proton movements to ATP formation in bacteria.

Maloney, P.C.; Hansen, F.C.

1982-01-01

333

Xylo-oligosaccharides and lactitol promote the growth of Bifidobacterium lactis and Lactobacillus species in pure cultures.  

PubMed

The current screening study aimed at identifying promising prebiotic and synbiotic candidates. The fermentation of xylo-oligosaccharides, xylan, galacto-oligosaccharide, fructo-oligosaccharide, polydextrose, lactitol, gentiobiose and pullulan was investigated in vitro. The ability of these established and potential prebiotic candidates to function as a sole carbon source for probiotic (Bifidobacterium and Lactobacillus), intestinal and potential pathogenic microbes (Eubacterium, Bacteroides, Clostridium, Escherichia coli, Salmonella, and Staphylococcus) was assessed in pure cultures. Xylo-oligosaccharides were fermented with high specificity by the tested Bifidobacterium lactis strains and lactitol by lactobacilli, whereas galacto-oligosaccharides, fructo-oligosaccharides and gentiobiose were utilised by a larger group of microbes. Xylan, polydextrose and pullulan were utilised to a limited extent by only a few of the tested microbes. The results of this screening study indicate that xylo-oligosaccharides and lactitol support the growth of a limited number of beneficial microbes in pure cultures. Such a high degree of specificity has not been previously reported for established prebiotics. Based on these results, the most promising prebiotics and synbiotic combinations can be selected for further testing. PMID:21840802

Mkelinen, H; Saarinen, M; Stowell, J; Rautonen, N; Ouwehand, A C

2010-06-01

334

The Maltose ABC Transporter in Lactococcus lactis Facilitates High-Level Sensitivity to the Circular Bacteriocin Garvicin ML  

PubMed Central

We generated and characterized a series of spontaneous mutants of Lactococcus lactis IL1403 with average 6- to 11-fold-lowered sensitivities to the circular bacteriocin garvicin ML (GarML). Carbohydrate fermentation assays highlighted changes in carbohydrate metabolism, specifically loss of the ability to metabolize starch and maltose, in these mutants. PCR and sequencing showed that a 13.5-kb chromosomal deletion encompassing 12 open reading frames, mainly involved in starch and maltose utilization, had spontaneously occurred in the GarML-resistant mutants. Growth experiments revealed a correlation between sensitivity to GarML and carbon catabolite repression (CCR); i.e., sensitivity to GarML increased significantly when wild-type cells were grown on maltose and galactose as sole carbohydrates, an effect which was alleviated by the presence of glucose. Among the genes deleted in the mutants were malEFG, which encode a CCR-regulated membrane-bound maltose ABC transporter. The complementation of mutants with these three genes recovered normal sensitivity to the bacteriocin, suggesting an essential role of the maltose ABC transporter in the antimicrobial activity of GarML. This notion was supported by the fact that the level of sensitivity to GarML was dose dependent, increasing with higher expression levels of malEFG over a 50-fold range. To our knowledge, this is the first time a specific protein complex has been demonstrated to be involved in sensitivity to a circular bacteriocin. PMID:22411612

Gabrielsen, Christina; Brede, Dag A.; Hernndez, Pablo E.; Nes, Ingolf F.

2012-01-01

335

An alkali-thermostable xylanase from Bacillus pumilus functionally expressed in Kluyveromyces lactis and evaluation of its deinking efficiency.  

PubMed

This work aimed at studying the recombinant expression of an alkali- and thermo-stable xylanase from Bacillus pumilus in Kluyveromyces lactis and its use in deinking of civic paper waste. Efficient expression with a 3-fold increase in the activity than the native organism was achieved. An inducer concentration of 2.5% and medium pH of 9.0 was the best for enzyme expression. Purified enzyme showed an optimum activity at temperatures 50 and 60C and pH 9.0 and 10.0, respectively. At pH 12.0, enzyme retained 74% and 26% activity after 2 and 3h of incubation, respectively. After incubation at 50 and 60C for 1h, the enzyme showed 100% retention of activity, and remained active for 4h at 60C retaining 23% residual activity. Partially purified recombinant enzyme showed higher deinking efficiency (273%) of laser print waste paper than crude xylanase from Bacillus and commercial acidic enzyme. This xylanase with superior stability characteristics could be a suitable candidate in paper and pulp industries. PMID:24709528

Thomas, Leya; Ushasree, Mrudula V; Pandey, Ashok

2014-08-01

336

Receptor-Binding Protein of Lactococcus lactis Phages: Identification and Characterization of the Saccharide Receptor-Binding Site  

PubMed Central

Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85-89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7- resolution structure of RBP, was found to bind tightly (Kd = 0.26 ?M) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain. PMID:16547026

Tremblay, Denise M.; Tegoni, Mariella; Spinelli, Silvia; Campanacci, Valrie; Blangy, Stphanie; Huyghe, Cline; Desmyter, Aline; Labrie, Steve; Moineau, Sylvain; Cambillau, Christian

2006-01-01

337

Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1.  

PubMed

In lactic acid bacteria, pentoses are metabolized via the phosphoketolase pathway, which catalyzes the cleavage of D-xylulose-5-phosphate to equimolar amounts of glyceraldehyde 3-phosphate and acetylphosphate. Hence the yield coefficient of lactate from pentose does not exceed 1.0 mol/mol, while that of Lactococcus lactis IO-1(JCM7638) at high D-xylose concentrations often exceeds the theoretical value. This suggests that, in addition to the phosphoketolase pathway, L. lactisIO-1 may possess another metabolic pathway that produces only lactic acid from xylose. In the present study, the metabolism of xylose in L. lactisIO-1 was deduced from the product formation and enzyme activities of L. lactisIO-1 in batch culture and continuous culture. During cultivation with xylose concentrations above ca. 50 g/l, the yield coefficient of L-lactate exceeded 1.0 mol/mol while those of acetate, formate and ethanol were very low. At xylose concentrations less than 5 g/l, acetate, formate and ethanol were produced with yield coefficients of about 1.0 mol/mol, while L-lactate was scarcely produced. In cells grown at high xylose concentrations, a marked decrease in the specific activities of phosphoketolase and pyruvate formate lyase (PFL), and an increase in those of transketolase and transaldolase were observed. These results indicate that in L. lactisIO-1 xylose may be catabolized by two different pathways, the phosphoketolase pathway yielding acetate, formate and ethanol, and the pentose phosphate (PP)/glycolytic pathway which converts xylose to L-lactate only. Furthermore, it was deduced that the change in the xylose concentration in the culture medium shifts xylulose 5-phosphate metabolism between the phosphoketolase pathway and the PP/glycolytic pathway in L. lactisIO-1, and pyruvate metabolism between cleavage to acetyl-CoA and formic acid by PFL and the reduction to L-lactate by lactate dehydrogenase. PMID:12382058

Tanaka, K; Komiyama, A; Sonomoto, K; Ishizaki, A; Hall, S J; Stanbury, P F

2002-10-01

338

RAS/Cyclic AMP and Transcription Factor Msn2 Regulate Mating and Mating-Type Switching in the Yeast Kluyveromyces lactis ?  

PubMed Central

In response to harsh environmental conditions, ascomycetes produce stress-resistant spores to promote survival. As sporulation requires a diploid DNA content, species with a haploid lifestyle, such as Kluyveromyces lactis, first induce mating in response to stress. In K. lactis, mating and mating-type switching are induced by the DNA-binding protein Mts1. Mts1 expression is known to be upregulated by nutrient limitation, but the mechanism is unknown. We show that a ras2 mutation results in a hyperswitching phenotype. In contrast, strains lacking the phosphodiesterase Pde2 had lower switching rates compared to that of the wild type (WT). As Ras2 promotes cyclic AMP (cAMP) production and Pde2 degrades cAMP, these data suggest that low cAMP levels induce switching. Because the MTS1 regulatory region contains several Msn2 binding sites and Msn2 is a transcription factor that is activated by low cAMP levels, we investigated if Msn2 regulates MTS1 transcription. Consistently with this idea, an msn2 mutant strain displayed lower switching rates than the WT strain. The transcription of MTS1 is highly induced in the ras2 mutant strain. In contrast, an msn2 ras2 double mutant strain displays WT levels of the MTS1 transcript, showing that Msn2 is a critical inducer of MTS1 transcription. Strains lacking Msn2 and Pde2 also exhibit mating defects that can be complemented by the ectopic expression of Mts1. Finally, we show that MTS1 is subjected to negative autoregulation, presumably adding robustness to the mating and switching responses. We suggest a model in which Ras2/cAMP/Msn2 mediates the stress-induced mating and mating-type switching responses in K. lactis. PMID:21890818

Barsoum, E.; Rajaei, N.; strm, S. U.

2011-01-01

339

Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-producing starter culture.  

PubMed

After isolation from different doughs and sourdoughs, 177 strains of lactic acid bacteria were screened at the phenotypic level for exopolysaccharide production on media containing different carbohydrate sources. Two exopolysaccharide-producing lactic acid bacteria (Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A) were selected through quantitative analysis on solid media containing sucrose and yeast extract. The PCR detection of homopolysaccharide (gtf and lev) and heteropolysaccharide (epsA, epsB, epsD and epsE, and epsEFG) genes showed different distributions within species and strains of the lactic acid bacteria studied. Moreover, in some strains both homopolysaccharide and heteropolysaccharide genes were detected. Proton nuclear magnetic resonance spectra suggest that Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A produced the same exopolysaccharide, which was constituted by a single repeating glucopyranosyl unit linked by an ?-(1?6) glycosidic bond in a dextran-type carbohydrate. Microbial growth, acidification, and viscoelastic properties of sourdoughs obtained by exopolysaccharide-producing and nonproducing lactic acid bacterial strains were evaluated. Sourdough obtained after 15 h at 30C with exopolysaccharide-producing lactic acid bacteria reached higher total titratable acidity as well as elastic and dissipative modulus curves with respect to the starter not producing exopolysaccharide, but they showed similar levels of pH and microbial growth. On increasing the fermentation time, no difference in the viscoelastic properties of exopolysaccharide-producing and nonproducing samples was observed. This study suggests that dextran-producing Leuconostoc lactis 95A and Lactobacillus curvatus 69B2 can be employed to prepare sourdough, and this would be particularly useful to improve the quality of baked goods while avoiding the use of commercially available hydrocolloids as texturizing additives. PMID:22307283

Palomba, Simona; Cavella, Silvana; Torrieri, Elena; Piccolo, Alessandro; Mazzei, Pierluigi; Blaiotta, Giuseppe; Ventorino, Valeria; Pepe, Olimpia

2012-04-01

340

Dietary supplementation with Bifidobacterium lactis NCC2818 from weaning reduces local immunoglobulin production in lymphoid-associated tissues but increases systemic antibodies in healthy neonates.  

PubMed

Weaning is associated with a major shift in the microbial community of the intestine, and this instability may make it more acquiescent than the adult microbiota to long-term changes. Modulation achieved through dietary interventions may have potentially beneficial effects on the developing immune system, which is driven primarily by the microbiota. The specific aim of the present study was to determine whether immune development could be modified by dietary supplementation with the human probiotic Bifidobacterium lactis NCC2818 in a tractable model of weaning in infants. Piglets were reared by their mothers before being weaned onto a solid diet supplemented with B. lactis NCC2818, while sibling controls did not receive supplementation. Probiotic supplementation resulted in a reduction in IgA (P<00005) and IgM (P<0009) production by mucosal tissues but had no effect on IgG production (P>005). Probiotic-supplemented pigs had more mast cells than unsupplemented littermates (P<00001), although numbers in both groups were low. In addition, the supplemented piglets made stronger serum IgG responses to fed and injected antigens (P<005). The present findings are consistent with B. lactis NCC2818 reducing intestinal permeability induced by weaning, and suggest that the piglet is a valuable intermediate between rodent models and human infants. The results also strongly suggest that measures of the effect of probiotic supplementation on the immune system need to be interpreted carefully as proxy measures of health benefit. However, they are useful in developing an understanding of the mechanism of action of probiotic strains, an important factor in predicting favourable health outcomes of nutritional intervention. PMID:23473077

Lewis, Marie C; Patel, Dilip V; Fowler, Jenni; Duncker, Swantje; Zuercher, Adrian W; Mercenier, Annick; Bailey, Mick

2013-10-01

341

Oxygen-Dependent Transcriptional Regulator Hap1p Limits Glucose Uptake by Repressing the Expression of the Major Glucose Transporter Gene RAG1 in Kluyveromyces lactis?  

PubMed Central

The HAP1 (CYP1) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae, Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1-equivalent gene (KlHAP1) in K. lactis. KlHap1p showed a number of sequence features and some gene targets (such as KlCYC1) in common with its S. cerevisiae counterpart, and KlHAP1 was capable of complementing the hap1 mutation. However, the KlHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The ?Klhap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis. PMID:18806211

Bao, Wei-Guo; Guiard, Bernard; Fang, Zi-An; Donnini, Claudia; Gervais, Michel; Passos, Flavia M. Lopes; Ferrero, Iliana; Fukuhara, Hiroshi; Bolotin-Fukuhara, Monique

2008-01-01

342

Polyphasic Screening, Homopolysaccharide Composition, and Viscoelastic Behavior of Wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus Exopolysaccharide-Producing Starter Culture  

PubMed Central

After isolation from different doughs and sourdoughs, 177 strains of lactic acid bacteria were screened at the phenotypic level for exopolysaccharide production on media containing different carbohydrate sources. Two exopolysaccharide-producing lactic acid bacteria (Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A) were selected through quantitative analysis on solid media containing sucrose and yeast extract. The PCR detection of homopolysaccharide (gtf and lev) and heteropolysaccharide (epsA, epsB, epsD and epsE, and epsEFG) genes showed different distributions within species and strains of the lactic acid bacteria studied. Moreover, in some strains both homopolysaccharide and heteropolysaccharide genes were detected. Proton nuclear magnetic resonance spectra suggest that Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A produced the same exopolysaccharide, which was constituted by a single repeating glucopyranosyl unit linked by an ?-(1?6) glycosidic bond in a dextran-type carbohydrate. Microbial growth, acidification, and viscoelastic properties of sourdoughs obtained by exopolysaccharide-producing and nonproducing lactic acid bacterial strains were evaluated. Sourdough obtained after 15 h at 30C with exopolysaccharide-producing lactic acid bacteria reached higher total titratable acidity as well as elastic and dissipative modulus curves with respect to the starter not producing exopolysaccharide, but they showed similar levels of pH and microbial growth. On increasing the fermentation time, no difference in the viscoelastic properties of exopolysaccharide-producing and nonproducing samples was observed. This study suggests that dextran-producing Leuconostoc lactis 95A and Lactobacillus curvatus 69B2 can be employed to prepare sourdough, and this would be particularly useful to improve the quality of baked goods while avoiding the use of commercially available hydrocolloids as texturizing additives. PMID:22307283

Palomba, Simona; Cavella, Silvana; Torrieri, Elena; Piccolo, Alessandro; Mazzei, Pierluigi; Blaiotta, Giuseppe; Ventorino, Valeria

2012-01-01

343

Development of a rapid SNP-typing assay to differentiate Bifidobacterium animalis ssp. lactis strains used in probiotic-supplemented dairy products.  

PubMed

Identification at the genus, species, and strain levels is desirable when a probiotic microorganism is added to foods. Strains of Bifidobacterium animalis ssp. lactis (BAL) are commonly used worldwide in dairy products supplemented with probiotic strains. However, strain discrimination is difficult because of the high degree of genome identity (99.975%) between different genomes of this subspecies. Typing of monomorphic species can be carried out efficiently by targeting informative single nucleotide polymorphisms (SNP). Findings from a previous study analyzing both reference and commercial strains of BAL identified SNP that could be used to discriminate common strains into 8 groups. This paper describes development of a minisequencing assay based on the primer extension reaction (PER) targeting multiple SNP that can allow strain differentiation of BAL. Based on previous data, 6 informative SNP were selected for further testing, and a multiplex preliminary PCR was optimized to amplify the DNA regions containing the selected SNP. Extension primers (EP) annealing immediately adjacent to the selected SNP were developed and tested in simplex and multiplex PER to evaluate their performance. Twenty-five strains belonging to 9 distinct genomic clusters of B. animalis ssp. lactis were selected and analyzed using the developed minisequencing assay, simultaneously targeting the 6 selected SNP. Fragment analysis was subsequently carried out in duplicate and demonstrated that the assay yielded 8 specific profiles separating the most commonly used commercial strains. This novel multiplex PER approach provides a simple, rapid, flexible SNP-based subtyping method for proper characterization and identification of commercial probiotic strains of BAL from fermented dairy products. To assess the usefulness of this method, DNA was extracted from yogurt manufactured with and without the addition of B. animalis ssp. lactis BB-12. Extracted DNA was then subjected to the minisequencing protocol, resulting in a SNP profile matching the profile for the strain BB-12. PMID:25547309

Lomonaco, Sara; Furumoto, Emily J; Loquasto, Joseph R; Morra, Patrizia; Grassi, Ausilia; Roberts, Robert F

2015-02-01

344

Use of non-growing Lactococcus lactis cell suspensions for production of volatile metabolites with direct relevance for flavour formation during dairy fermentations.  

PubMed

Background Lactococcus lactis is a lactic acid bacterium that has been used for centuries in the production of a variety of cheeses, as these bacteria rapidly acidify milk and greatly contribute to the flavour of the fermentation end-products. After a short growth phase during cheese ripening L. lactis enters an extended non-growing state whilst still strongly contributing to amino acid-derived flavour formation. Here, a research approach is presented that allows investigation of strain- and amino acid-specific flavour formation during the non-growing state.ResultsNon-growing cells of five selected L. lactis strains were demonstrated to degrade amino acids into flavour compounds that are relevant in food fermentations and differs greatly from production of flavour compounds using growing cells. As observed earlier in other research set-ups and with other microorganisms, addition of NADH, -ketoglutarate and pyridoxal-5-phosphate was demonstrated to be essential for optimal flavour formation, suggesting that intracellular pools of these substrates are too low for the significant production of the flavour compounds. Production of flavours during the non-growing phase strongly depends on the individual amino acids that were supplied, on the presence of other amino acids (mixtures versus single compounds), and on the strain used. Moreover, we observed that the plasmid-free model strains L. lactis MG1363 and IL1403 produce relatively low amounts of flavour components under the various conditions tested.ConclusionsBy using this simplified and rapid approach to study flavour formation by non-growing lactic acid bacteria, lengthy ripening periods are no longer required to assess the capacity of strains to produce flavours in the long, non-growing state of dairy fermentation. In addition, this method also provides insight into the conversion of single amino acids versus the conversion of a mixture of amino acids as produced during protein degradation. The generated results are complementary to earlier generated datasets using growing cells, allowing assessment of the full flavour forming potential of strains used as starter cultures in industrial food fermentation processes. PMID:25492249

van de Bunt, Bert; Bron, Peter A; Sijtsma, Lolke; de Vos, Willem M; Hugenholtz, Jeroen

2014-12-10

345

Influence of intracellular pH on light emission from a luxA/B derivative of Lactococcus lactis subsp. diacetylactis.  

PubMed

High levels of constitutive aldehyde-dependent light emission were obtained from nongrowing cells of Lactococcus lactis subsp. diacetylactis F712 transformed with IuxA/B when they were suspended in buffered solutions. Inductions of light emission was time-dependent and was not due to growth, synthesis of luciferase or stimulation of metabolism by fermentable carbohydrate. The major factor controlling light emission in such cells appears to be the intracellular pH value. Experiments with ionophores indicated that a transmembrane pH gradient was not essential for light emission. PMID:8493883

Simpson, W J

1993-01-01

346

Technological characterization and survival of the exopolysaccharide-producing strain Lactobacillus delbrueckii subsp. lactis 193 and its bile-resistant derivative 193+ in simulated gastric and intestinal juices.  

PubMed

The capacity of lactic acid bacteria to produce exopolysaccharides (EPS) conferring microorganisms a ropy phenotype could be an interesting feature from a technological point of view. Progressive adaptation to bile salts might render some lactobacilli able to overcome physiological gut barriers but could also modify functional properties of the strain, including the production of EPS. In this work some technological properties and the survival ability in simulated gastrointestinal conditions of Lactobacillus delbrueckii subsp. lactis 193, and Lb. delbrueckii subsp. lactis 193+, a strain with stable bile-resistant phenotype derived thereof, were characterized in milk in order to know whether the acquisition of resistance to bile could modify some characteristics of the microorganism. Both strains were able to grow and acidify milk similarly; however the production of ethanol increased at the expense of the aroma compound acetaldehyde in milk fermented by the strain 193+, with respect to milk fermented by the strain 193. Both microorganisms produced a heteropolysaccharide composed of glucose and galactose, and were able to increase the viscosity of fermented milks. In spite of the higher production yield of EPS by the bile-resistant strain 193+, it displayed a lower ability to increase viscosity than Lb. delbrueckii subsp. lactis 193. Milk increased survival in simulated gastric juice; the presence of bile improved adhesion to the intestinal cell line HT29-MTX in both strains. However, the acquisition of a stable resistance phenotype did not improve survival in simulated gastric and intestinal conditions or the adhesion to the intestinal cell line HT29-MTX. Thus, Lb. delbrueckii subsp. lactis 193 presents suitable technological properties for the manufacture of fermented dairy products; the acquisition of a stable bile-resistant phenotype modified some properties of the microorganism. This suggests that the possible use of bile-resistant derivative strains should be carefully evaluated in each specific application considering the influence that the acquisition of a stable bile-resistant phenotype could have in survival ability in gastric and intestinal conditions and in technological properties. PMID:21774862

Burns, Patricia; Vinderola, Gabriel; Reinheimer, Jorge; Cuesta, Isabel; de Los Reyes-Gaviln, Clara G; Ruas-Madiedo, Patricia

2011-08-01

347

Influence of cofermentation by amylolytic Lactobacillus plantarum and Lactococcus lactis strains on the fermentation process and rheology of sorghum porridge.  

PubMed

Amylolytic lactic acid bacteria (ALAB) can potentially replace malt in reducing the viscosity of starchy porridges. However, the drawback of using ALAB is their low and delayed amylolytic activity. This necessitates searching for efficient ALAB and strategies to improve their amylolytic activity. Two ALAB, Lactobacillus plantarum MNC 21 and Lactococcus lactis MNC 24, isolated from Obushera, were used to ferment starches in MRS broth: sorghum, millet, sweet potato, and commercial soluble starch. The amylolytic activity of MNC 21 was comparable to that of the ALAB collection strain Lb. plantarum A6, while that of MNC 24 was extremely low. MNC 21, MNC 24, and their coculture were compared to A6 and sorghum malt for ability to ferment and reduce the viscosity of sorghum porridge (11.6% dry matter). ALAB and the coculture lowered the pH from 6.2 to <4.5 within 12 h, while malt as a carrier of wild starter took about 20 h. Coculturing increased lactic acid yield by 46% and 76.8% compared to the yields of MNC 21 and MNC 24 monocultures, respectively. The coculture accumulated significantly larger (P < 0.05) amounts of maltose and diacetyl than the monocultures. Sorghum malt control and the coculture hydrolyzed more starch in sorghum porridge than the monocultures. The coculture initiated changes in the rheological parameters storage modulus (G'), loss modulus (G?), phase angle (?), and complex viscosity (?*) earlier than its constituent monocultures. The shear viscosity of sorghum porridge was reduced significantly (P < 0.05) from 1950 cP to 110 cP (malt), 281 cP (coculture), 382 cP (MNC 21), 713 cP (MNC 24), and 722 cP (A6). Coculturing strong ALAB with weak ALAB or non-ALAB can be exploited for preparation of nutrient-dense weaning foods and increasing lactic acid yield from starchy materials. PMID:22610432

Mukisa, Ivan M; Byaruhanga, Yusuf B; Muyanja, Charles M B K; Aijuka, Matthew; Schller, Reidar B; Sahlstrm, Stefan; Langsrud, Thor; Narvhus, Judith A

2012-08-01

348

Enhancement of Ad-CRT/E7-mediated antitumor effect by preimmunization with L. lactis expressing HPV-16 E7.  

PubMed

Although current polyvalent vaccines can prevent development of cervical cancer, they cannot be used to treat patients who already have the disease. Adenovirus expressing calreticulin-E7 (Ad-CRT-E7) has shown promising results in the cervical cancer murine model. We also demonstrated that immunization with Lactococcus lactis encoding HPV-16 E7 (Ll-E7) anchored to its surface induces significant HPV-16 E7-specific immune response. Here, we assessed the combination of both approaches in the treatment of a cervical cancer animal model. Intranasal preimmunization of Ll-E7, followed by a single Ad-CRT/E7 application, induced ?80% of tumor suppression in comparison with controls. Mice treated with a combination of Ll-E7 and Ad-CRT/E7 resulted in a 70% survival rate 300 days post-treatment, whereas 100% of the mice in the control groups died by 50 days. Significant CD8+ cytotoxic T-lymphocytes infiltration was detected in the tumors of mice treated with Ll-E7+Ad-CRT/E7. Tumors with regression showed a greater number of positive cells for in situ TUNEL staining than controls. Our results suggest that preimmunization with Ll-E7 enhances the Ad-CRT/E7-mediated antitumor effect. This treatment provides an enormous advantage over repeated applications of Ad-CRT/E7 by maintaining the effectiveness of the three-dose application of Ad-CRT/E7, but avoiding the high systemic toxicities associated with such repeat treatments. PMID:25216057

Rangel-Colmenero, Blanca R; Gomez-Gutierrez, Jorge G; Villatoro-Hernndez, Julio; Zavala-Flores, Laura M; Quistin-Martnez, Deyanira; Rojas-Martnez, Augusto; Arce-Mendoza, Alma Y; Guzmn-Lpez, Santos; Montes-de-Oca-Luna, Roberto; Saucedo-Crdenas, Odila

2014-11-01

349

Influence of Cofermentation by Amylolytic Lactobacillus plantarum and Lactococcus lactis Strains on the Fermentation Process and Rheology of Sorghum Porridge  

PubMed Central

Amylolytic lactic acid bacteria (ALAB) can potentially replace malt in reducing the viscosity of starchy porridges. However, the drawback of using ALAB is their low and delayed amylolytic activity. This necessitates searching for efficient ALAB and strategies to improve their amylolytic activity. Two ALAB, Lactobacillus plantarum MNC 21 and Lactococcus lactis MNC 24, isolated from Obushera, were used to ferment starches in MRS broth: sorghum, millet, sweet potato, and commercial soluble starch. The amylolytic activity of MNC 21 was comparable to that of the ALAB collection strain Lb. plantarum A6, while that of MNC 24 was extremely low. MNC 21, MNC 24, and their coculture were compared to A6 and sorghum malt for ability to ferment and reduce the viscosity of sorghum porridge (11.6% dry matter). ALAB and the coculture lowered the pH from 6.2 to <4.5 within 12 h, while malt as a carrier of wild starter took about 20 h. Coculturing increased lactic acid yield by 46% and 76.8% compared to the yields of MNC 21 and MNC 24 monocultures, respectively. The coculture accumulated significantly larger (P < 0.05) amounts of maltose and diacetyl than the monocultures. Sorghum malt control and the coculture hydrolyzed more starch in sorghum porridge than the monocultures. The coculture initiated changes in the rheological parameters storage modulus (G?), loss modulus (G?), phase angle (?), and complex viscosity (?*) earlier than its constituent monocultures. The shear viscosity of sorghum porridge was reduced significantly (P < 0.05) from 1950 cP to 110 cP (malt), 281 cP (coculture), 382 cP (MNC 21), 713 cP (MNC 24), and 722 cP (A6). Coculturing strong ALAB with weak ALAB or non-ALAB can be exploited for preparation of nutrient-dense weaning foods and increasing lactic acid yield from starchy materials. PMID:22610432

Byaruhanga, Yusuf B.; Muyanja, Charles M. B. K.; Aijuka, Matthew; Schller, Reidar B.; Sahlstrm, Stefan; Langsrud, Thor; Narvhus, Judith A.

2012-01-01

350

Growth performance of early-weaned pigs is enhanced by feeding epidermal growth factor-expressing Lactococcus lactis fermentation product.  

PubMed

We have previously generated epidermal growth factor expressing Lactococcus lactis (EGF-LL) using bioengineering approach, and shown that feeding newly-weaned piglets EGF-LL improves digestive function. To address concerns over the use of genetically modified organisms (GMO), the objective of the current study was to investigate the effect of feeding the EGF-LL fermentation product, after removal of the genetically modified EGF-LL, on growth performance and intestine development of newly-weaned piglets. One hundred and twenty newly-weaned piglets were fed ad libitum according to a 2-phase feeding program. Four pens were assigned to each of three treatments: (1) complete EGF-LL fermentation product (Ferm), (2) supernatant of EGF-LL fermentation product, after removal of EGF-LL (Supern), or (3) blank M17GE media (Control). EGF-LL or its fermented supernatant was administrated to piglets in the first 3 weeks post-weaning; their growth performance was monitored throughout treatment, and for the following week. Daily body weight gain (254.8g vs. 200.5g) and Gain:Feed (0.541kg/kg vs. 0.454kg/kg) of pigs on the Supern group were significantly improved compared to that of Control, although no difference was observed between the Ferm and Control pigs. Intestinal sucrase activity was increased in Supern- compared to Control group (166.362.1 vs. 81.456.5nmol glucose released/mg protein; P<0.05). The lack of growth response with Ferm pigs may be attributed to an overload of bacteria (daily dose included 4.5610(10)CFU/kg BW/day EGF-LL). These results suggest that GMO-free EGF-LL fermentation product is effective in increasing growth performance of early-weaned piglets. PMID:24445174

Bedford, Andrea; Huynh, Evanna; Fu, Molei; Zhu, Cuilan; Wey, Doug; de Lange, Cornelis; Li, Julang

2014-03-10

351

Mutation in the beta subunit of F ATPase allows Kluyveromyces lactis to survive the disruption of the KlPGS1 gene.  

PubMed

The petite-negative yeast Kluyveromyces lactis does not tolerate the loss of phosphatidylglycerol (PG). We demonstrate that the lethality of PG loss is suppressed in strains carrying a mutation in the beta subunit of F(1) ATPase (mgi1-1). Phenotypic characterization shows that the strain lacking the phosphatidylglycerolphosphate synthase gene (KlPGS1) is able to grow only on glucose, but significantly more slowly and to substantially lower densities than the parental mgi1-1 strain. In addition, oxygen consumption in the DeltaKlpgs1 strain is <1% of the parental strain. Western blot analysis of mitochondrial membrane proteins shows that the amounts of some proteins are substantially decreased or even not detectable in this mutant. However, overexpression of the KlPGS1 gene under the inducible GAL1 promoter does not restore the ability of DeltaKlpgs1 cells to grow on galactose, indicating the presence of some other mutations and/or deletions in genes involved in oxidative phosphorylation. We also demonstrate that DeltaKlpgs1 cells do not spontaneously lose mtDNA, but are able to survive its loss after ethidium bromide mutagenesis. Deletion of the cardiolipin synthase gene (KlCLS1) in mgi1-1 has only a minimal effect on mitochondrial physiology, and additional experiments show that this deletion is also viable in wild-type K. lactis. PMID:20528952

Patrsov, Mria; Kost'anov-Poliakov, Daniela; Simockov, Mria; Sabov, L'udmila

2010-09-01

352

Structure and Function of CinD (YtjD) of Lactococcus lactis, a Copper-Induced Nitroreductase Involved in Defense against Oxidative Stress ?  

PubMed Central

In Lactococcus lactis IL1403, 14 genes are under the control of the copper-inducible CopR repressor. This so-called CopR regulon encompasses the CopR regulator, two putative CPx-type copper ATPases, a copper chaperone, and 10 additional genes of unknown function. We addressed here the function of one of these genes, ytjD, which we renamed cinD (copper-induced nitroreductase). Copper, cadmium, and silver induced cinD in vivo, as shown by real-time quantitative PCR. A knockout mutant of cinD was more sensitive to oxidative stress exerted by 4-nitroquinoline-N-oxide and copper. Purified CinD is a flavoprotein and reduced 2,6-dichlorophenolindophenol and 4-nitroquinoline-N-oxide with kcat values of 27 and 11 s?1, respectively, using NADH as a reductant. CinD also exhibited significant catalase activity in vitro. The X-ray structure of CinD was resolved at 1.35 and resembles those of other nitroreductases. CinD is thus a nitroreductase which can protect L. lactis against oxidative stress that could be exerted by nitroaromatic compounds and copper. PMID:20562311

Mermod, Mlanie; Mourlane, Frdric; Waltersperger, Sandro; Oberholzer, Anselm E.; Baumann, Ulrich; Solioz, Marc

2010-01-01

353

Effect of the Abortive Infection Mechanism and Type III Toxin/Antitoxin System AbiQ on the Lytic Cycle of Lactococcus lactis Phages  

PubMed Central

To survive in phage-containing environments, bacteria have evolved an array of antiphage systems. Similarly, phages have overcome these hurdles through various means. Here, we investigated how phages are able to circumvent the Lactococcus lactis AbiQ system, a type III toxin-antitoxin with antiviral activities. Lactococcal phage escape mutants were obtained in the laboratory, and their genomes were sequenced. Three unrelated genes of unknown function were mutated in derivatives of three distinct lactococcal siphophages: orf38 of phage P008, m1 of phage bIL170, and e19 of phage c2. One-step growth curve experiments revealed that the phage mutations had a fitness cost while transcriptional analyses showed that AbiQ modified the early-expressed phage mRNA profiles. The L. lactis AbiQ system was also transferred into Escherichia coli MG1655 and tested against several coliphages. While AbiQ was efficient against phages T4 (Myoviridae) and T5 (Siphoviridae), escape mutants of only phage 2 (Myoviridae) could be isolated. Genome sequencing revealed a mutation in gene orf210, a putative DNA polymerase. Taking these observations together, different phage genes or gene products are targeted or involved in the AbiQ phenotype. Moreover, this antiviral system is active against various phage families infecting Gram-positive and Gram-negative bacteria. A model for the mode of action of AbiQ is proposed. PMID:23813728

Samson, Julie E.; Blanger, Maxime

2013-01-01

354

Microbial production of palatinose through extracellular expression of a sucrose isomerase from Enterobacter sp. FMB-1 in Lactococcus lactis MG1363.  

PubMed

Sucrose isomerase (SIase) has been used to produce palatinose, a structural isomer of sucrose, which has many beneficial health properties, such as low-glycemic and low-insulinemic indices. A gene corresponding to SIase from Enterobacter sp. FMB-1 was expressed in Lactococcus lactis MG1363 using the P170 expression system. The autoinducible promoter (P170) and an optimized signal peptide (SP310mut2) were used to induce and secrete SIase in L. lactis. One-step Ni-NTA affinity chromatography and Western blot analysis demonstrated that SIase was successfully secreted to the culture supernatant, although 60% of the recombinant enzymes were retained inside the cells. The production of the recombinant SIase was highly correlated with pH (pH 6) and glucose concentration (30g/L) of the medium. The extracellularly produced recombinant SIase was functionally active, effectively transforming 50g/L sucrose to 36g/L palatinose, with a conversion rate of 72% in the culture supernatant. PMID:20620050

Park, Jong-Yul; Jung, Jong-Hyun; Seo, Dong-Ho; Ha, Suk-Jin; Yoon, Jong-Won; Kim, Young-Cheul; Shim, Jae-Hoon; Park, Cheon-Seok

2010-11-01

355

A maturation protein is essential for production of active forms of Lactococcus lactis SK11 serine proteinase located in or secreted from the cell envelope.  

PubMed Central

The complete nucleotide sequence of a gene located immediately upstream of the Lactococcus lactis subsp. cremoris SK11 prtP gene encoding the cell envelope-attached proteinase was determined. This gene, designated prtM, was found to be transcribed from the same promotor region as was the proteinase gene but in the opposite direction. The prtM gene directed the expression in Escherichia coli of a protein with a size similar to the expected value of 33 kilodaltons, as deduced from the nucleotide sequence data. The derived amino acid sequence of the PrtM protein indicated the presence of a consensus lipoprotein signal sequence at the N terminus, which suggested that PrtM is a lipoprotein. Plasmids containing the prtM gene, the prtP gene, or both were constructed. Expression studies of L. lactis clones containing these plasmids showed that the prtM gene encodes a trans-acting activity involved in the maturation of cell envelope-located and -secreted forms of the SK11 proteinase. Images PMID:2496115

Vos, P; van Asseldonk, M; van Jeveren, F; Siezen, R; Simons, G; de Vos, W M

1989-01-01

356

Effects of Bifidobacterium lactis Bb12 Supplementation on Intestinal Microbiota of Preterm Infants: a Double-Blind, Placebo-Controlled, Randomized Study?  

PubMed Central

The gastrointestinal microbiota of preterm infants in a neonatal intensive care unit differs from that of term infants. In particular, the colonization of preterm infants by bifidobacteria is delayed. A double-blind, placebo-controlled, randomized clinical study was performed on 69 preterm infants to investigate the role of Bifidobacterium lactis Bb12 supplementation in modifying the gut microbiota. Both culture-dependent and culture-independent approaches were used to study the gut microbiota. Bifidobacterial numbers, determined by fluorescence in situ hybridization, were significantly higher in the probiotic than in the placebo group (log10 values per g of fecal wet weight: probiotic, 8.18 + 0.54 [standard error of the mean]; placebo, 4.82 + 0.51; P < 0.001). A similar trend for bifidobacterial numbers was also obtained with the culture-dependent method. The infants supplemented with Bb12 also had lower viable counts of Enterobacteriaceae (log10 values of CFU per g of fecal wet weight: probiotic, 7.80 + 0.34; placebo, 9.03 + 0.35; P = 0.015) and Clostridium spp. (probiotic, 4.89 + 0.30; placebo, 5.99 + 0.32; P = 0.014) than the infants in the placebo group. Supplementation of B. lactis Bb12 did not reduce the colonization by antibiotic-resistant organisms in the study population. However, the probiotic supplementation increased the cell counts of bifidobacteria and reduced the cell counts of enterobacteria and clostridia. PMID:16971641

Mohan, Ruchika; Koebnick, Corinna; Schildt, Janko; Schmidt, Sabine; Mueller, Manfred; Possner, Mike; Radke, Michael; Blaut, Michael

2006-01-01

357

BIOFIOBACTERIUM LACTIS ENHANCES TOLL-LIKE RECEPTOR (TLR) PATHWAY GENE EXPRESSION LOCALLY IN THE COLON AND ENHANCES GLUCOSE UPTAKE IN THE SMALL INTESTINE OF PIGS INFECTED WITH PARASITIC NEMATODE ASCARIS SUM.  

Technology Transfer Automated Retrieval System (TEKTRAN)

The addition of probiotic bacteria to the diet is proposed to enhance healthy responses to allergic and infectious diseases in pediatric populations, but quantitative data is often lacking. An experimental model was developed to inoculate neonatal pigs from birth with B. lactis (Bb12), detect relati...

358

Isolation and molecular characterization of KlCOX14, a gene of Kluyveromyces lactis encoding a protein necessary for the assembly of the cytochrome oxidase complex.  

PubMed

The yeast Kluyveromyces lactis was mutagenized with ethyl methane sulphonate and mutants unable to grow on respiratory carbon sources were isolated. Functional complementation of one of these mutants led to the isolation of KlCOX14, a gene encoding a 64 amino acid protein which is the functional homologue of Saccharomyces cerevisiae Cox14p, a protein necessary for the assembly of the cytochrome oxidase holoenzyme (Glerum et al., 1995). The disruption of KlCOX14 resulted in the absence of the absorption bands relative to cytochromes a and a(3) and in the complete loss of respiratory activity. Klcox14 mutants display the typical phenotype of pet mutants and have a reduced growth rate. In addition, unlike the wild-type, Klcox14 mutants are able to grow by fermentation also in the presence of low glucose. The nucleotide sequence of KlCOX14 has been deposited in the EMBL databank with Accession No. AJ238801. PMID:10669868

Fiori, A; Saliola, M; Goffrini, P; Falcone, C

2000-03-15

359

ScrFI restriction-modification system of Lactococcus lactis subsp. cremoris UC503: cloning and characterization of two ScrFI methylase genes.  

PubMed Central

Two genes from the total genomic DNA of dairy starter culture Lactococcus lactis subsp. cremoris UC503, encoding ScrFI modification enzymes, have been cloned and expressed in Escherichia coli. No homology between the two methylase genes was detected, and inverse polymerase chain reaction of flanking chromosomal DNA indicated that both were linked on the Lactococcus genome. Neither clone encoded the cognate endonuclease. The DNA sequence of one of the methylase genes (encoded by pCI931M) was determined and consisted of an open reading frame 1,170 bp long, which could encode a protein of 389 amino acids (M(r), 44.5). The amino acid sequence contained the highly characteristic motifs of an m5C methylase. Extensive regions of homology were observed with the methylases of NlaX, EcoRII, and Dcm. Images PMID:8481004

Davis, R; van der Lelie, D; Mercenier, A; Daly, C; Fitzgerald, G F

1993-01-01

360

Further studies on the glycerol teichoic acid of walls of Staphylococcus lactis I3. Location of the phosphodiester groups and their susceptibility to hydrolysis with alkali  

PubMed Central

1. The teichoic acid from walls of Staphylococcus lactis I3 is readily degraded in dilute alkali. 2. Degradation proceeds by selective hydrolysis of that phosphodiester group attached to an alcoholic hydroxyl group of the N-acetylglucosamine and gives a repeating unit in high yield. 3. Further studies on a different repeating unit isolated by partial acid hydrolysis have shown that the glycerol diphosphate is attached to the 4-hydroxyl group of the N-acetylglucosamine and not to the 3-hydroxyl group as was proposed earlier. 4. The susceptibility towards hydrolysis by alkali of other structural types of teichoic acid has been examined and found to vary markedly according to their structure. PMID:5158917

Archibald, A. R.; Baddiley, J.; Heckels, J. E.; Heptinstall, S.

1971-01-01

361

Backbone NMR resonance assignments of the nucleotide binding domain of the ABC multidrug transporter LmrA from Lactococcus lactis in its ADP-bound state.  

PubMed

LmrA from Lactococcus lactis is a multidrug transporter and a member of the ATP binding cassette (ABC) transporter family. ABC transporters consist of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). The NBD contains the highly conserved signature motifs of this transporter superfamily. In the case of LmrA, the TMD and the NBD are expressed as a single polypeptide. LmrA catalyzes the extrusion of hydrophobic compounds including antibiotics from the cell membrane at the expense of ATP hydrolysis. ATP binds to the NBD, where binding and hydrolysis induce conformational changes that lead to the extrusion of the substrate via the TMD. Here, we report the (1)H, (13)C and (15)N backbone chemical shift assignments of the isolated 263 amino acid containing NBD of LmrA in its ADP bound state. PMID:21786024

Hellmich, Ute A; Duchardt-Ferner, Elke; Glaubitz, Clemens; Whnert, Jens

2012-04-01

362

Isolation of Lactococcus lactis Mutants Simultaneously Resistant to the Cell Wall-Active Bacteriocin Lcn972, Lysozyme, Nisin, and Bacteriophage c2  

PubMed Central

Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits cell wall biosynthesis by binding to lipid II. In this work, two mutants resistant to Lcn972, Lactococcus lactis D1 and D1-20, with high (>320 arbitrary units [AU]/ml) and low (80 AU/ml) susceptibilities, respectively, have been isolated. Resistance to Lcn972 did not impose a burden to growth under laboratory conditions, nor did it substantially alter the physicochemical properties of the cell surface. However, the peptidoglycan of the mutants featured a higher content of muropeptides with tripeptide side chains than the wild-type strain, linking for the first time peptidoglycan remodelling to bacteriocin resistance. Moreover, L. lactis lacking a functional d,d-carboxypeptidase DacA (i.e., with a high content of pentapeptide side chain muropeptides) was shown to be more susceptible to Lcn972. Cross-resistance to lysozyme and nisin and enhanced susceptibility to penicillin G and bacitracin was also observed. Intriguingly, the Lcn972-resistant mutants were not infected by the lytic phage c2 and less efficiently infected by phage sk1. Lack of c2 infectivity was linked to a 22.6-kbp chromosomal deletion encompassing the phage receptor protein gene pip. The deletion also included maltose metabolic genes and the two-component system (TCS) F. However, a clear correlation between these genes and resistance to Lcn972 could not be clearly established, pointing to the presence of as-yet-unidentified mutations that account for Lcn972 resistance. PMID:22504807

Roces, Clara; Courtin, Pascal; Kulakauskas, Saulius; Rodrguez, Ana; Chapot-Chartier, Marie-Pierre

2012-01-01

363

Lactococcus lactis TrxD represents a subgroup of thioredoxins prevalent in Gram-positive bacteria containing WCXDC active site motifs.  

PubMed

Three protein disulfide reductases of the thioredoxin superfamily from the industrially important Gram-positive Lactococcus lactis (LlTrxA, LlTrxD and LlNrdH) are compared to the "classical" thioredoxin from Escherichia coli (EcTrx1). LlTrxA resembles EcTrx1 with a WCGPC active site motif and other key residues conserved. By contrast, LlTrxD is more distantly related and contains a WCGDC motif. Bioinformatics analysis suggests that LlTrxD represents a subgroup of thioredoxins from Gram-positive bacteria. LlNrdH is a glutaredoxin-like electron donor for ribonucleotide reductase class Ib. Based on protein-protein equilibria LlTrxA (E'=-259mV) and LlNrdH (E'=-238mV) show approximately 10mV higher standard state redox potentials than the corresponding E. coli homologues, while E' of LlTrxD is -243mV, more similar to glutaredoxin than "classical" thioredoxin. EcTrx1 and LlTrxA have high capacity to reduce insulin disulfides and their exposed active site thiol is alkylated at a similar rate at pH 7.0. LlTrxD on the other hand, is alkylated by iodoacetamide at almost 100 fold higher rate and shows no activity towards insulin disulfides. LlTrxA, LlTrxD and LlNrdH are all efficiently reduced by NADPH dependent thioredoxin reductase (TrxR) from L. lactis and good cross-reactivity towards E. coli TrxR was observed with LlTrxD as the notable exception. PMID:25255970

Bjrnberg, Olof; Efler, Petr; Ebong, Epie Denis; Svensson, Birte; Hgglund, Per

2014-12-15

364

Epidermal growth factor-expressing Lactococcus lactis enhances growth performance of early-weaned pigs fed diets devoid of blood plasma.  

PubMed

The effect of supplementing Lactococcus lactis (L. lactis) that was engineered to express epidermal growth factor (EGF-LL) to early-weaned pigs fed diets with typical levels of blood plasma (5%) or diets without blood plasma [blood plasma was substituted with soybean (Glycine max) meal and fish meal, based on amino acid supply] was examined. A total of 108 weaned piglets (19-26 d of age; mean initial BW 6.58 kg; 9 pigs per pen) were fed ad libitum according to a 2-phase feeding program without growth promoters. Three pens were assigned to each of 4 treatments: i) blood plasma-containing diet with blank bacterial growth medium (BP-Con), ii) blood plasma-containing diet with fermented EGF-LL (BP-EGF), iii) blood plasma-free diet with blank bacterial growth medium (BPF-Con), and iv) blood plasma-free diet with fermented EGF-LL (BPF-EGF). The amount of epidermal growth factor (EGF) was determined in the fermentation product and pigs were allotted 60 ?g EGF/kg BW/d for 3 wk postweaning. There were no differences in overall growth performance between BP-Con and BP-EGF pigs and no differences in overall growth performance between LoCon and BPF-EGF pigs. Pigs fed BPF-EGF showed increased daily BW gain (410 vs. 260 g/d; P < 0.01) and gain:feed (0.67 vs. 0.58; P < 0.05) compared to BPF-Con pigs in wk 3 postweaning; this was comparable to values for the BP-Con group (400 g/d and 0.64). These results indicate that supplementation with EGF-LL can be effective in enhancing the performance of early-weaned piglets fed a low complexity diet and reduces the need for feeding high-quality animal proteins and antibiotics. PMID:23365266

Bedford, A; Li, Z; Li, M; Ji, S; Liu, W; Huai, Y; de Lange, C F M; Li, J

2012-12-01

365

Comparative expression of lipase CAL-A in the yeasts Saccharomyces cerevisiae, Kluyveromyces lactis and Hansenula polymorpha to investigate a possible host influence.  

PubMed

Yeasts are an attractive expression platform, as they combine the ease of handling with the eukaryotic ability to process the produced protein. Important aspects of eukaryotic protein expression are posttranslational modifications, which can be required for functional expression of the protein of interest and can only be performed by eukaryotes. Each organism has its own modification pattern: for instance, the same protein produced in different hosts is subjected to various glycosylation pathways. It is amenable that these kinds of modifications can have an influence on the biochemical properties of the protein. To verify this thesis, the well-studied lipase CAL-A from Candida antarctica was chosen as a model enzyme. The codon bias of the gene sequence was uniformly optimized and expressed in three industrially relevant yeast hosts: Saccharomyces cerevisiae, Kluyveromyces lactis, and Hansenula polymorpha. The capacity of the expression systems to produce the enzyme was analyzed, as well as the biochemical properties of the produced and purified CAL-A. All hosts produced active enzyme; however, significant differences in the obtained yield were observed. H. polymorpha appeared to be the most productive host with a tenfold increase in productivity in comparison to S. cerevisiae. Studies on thermostability and activity of the purified enzymes towards various substrates showed a significant impact of the host on the biochemical properties of the produced protein. The most thermostable CAL-A from K. lactis retained 70% of its activity after incubation at 60C, in comparison to 45% remaining activity for the enzyme purified from S. cerevisiae. In the screenings with different substrates, a fourfold increase in activity between the enzymes from H. polymorpha and S. cerevisiae was found. Altogether, we herein exemplify how the selection of the host even within one taxonomic family (Saccharomycetaceae) significantly affects the produced enzyme's characteristics. PMID:25172438

Morka, Kamila; Pietruszka, Jrg; Meyer Zu Berstenhorst, Sonja

2014-12-10

366

The ltp gene of temperate Streptococcus thermophilus phage TP-J34 confers superinfection exclusion to Streptococcus thermophilus and Lactococcus lactis  

SciTech Connect

The ltp gene, located within the lysogeny module of temperate Streptococcus thermophilus phage TP-J34, has been shown to be expressed in lysogenic strain S. thermophilus J34. It codes for a lipoprotein, as demonstrated by inhibition of cleavage of the signal sequence by globomycin. Exposure of Ltp on the surface of Lactococcus lactis protoplasts bearing a plasmid-encoded copy of ltp has been demonstrated by immunogold labeling and electron microscopy. Expression of ltp in prophage- and plasmid-cured S. thermophilus J34-6f interfered with TP-J34 infection. While plating efficiency was reduced by a factor of about 40 and lysis of strain J34-6f in liquid medium was delayed considerably, phage adsorption was not affected at all. Intracellular accumulation of phage DNA was shown to be inhibited by Ltp. This indicates interference of Ltp with infection at the stage of triggering DNA release and injection into the cell, indicating a role of Ltp in superinfection exclusion. Expression of ltp in L. lactis Bu2-60 showed that the same superinfection exclusion mechanism was strongly effective against phage P008, a member of the lactococcal 936 phage species: no plaque-formation was detectable with even 10{sup 9} phage per ml applied, and lysis in liquid medium did not occur. In Lactococcus also, Ltp apparently inhibited phage DNA release and/or injection. Ltp appears to be a member of a family of small, secreted proteins with a 42 amino acids repeat structure encoded by genes of Gram-positive bacteria. Some of these homologous genes are part of the genomes of prophages.

Sun Xingmin [Institute for Microbiology, Federal Research Center for Nutrition and Food, PO Box 6069, D-24121 Kiel (Germany)]. E-mail: Xingmin_Sun@brown.edu; Goehler, Andre [Institute for Microbiology, Federal Research Center for Nutrition and Food, PO Box 6069, D-24121 Kiel (Germany); Heller, Knut J. [Institute for Microbiology, Federal Research Center for Nutrition and Food, PO Box 6069, D-24121 Kiel (Germany)]. E-mail: knut.heller@bfel.de; Neve, Horst [Institute for Microbiology, Federal Research Center for Nutrition and Food, PO Box 6069, D-24121 Kiel (Germany)

2006-06-20

367

L. plantarum, L. salivarius, and L. lactis Attenuate Th2 Responses and Increase Treg Frequencies in Healthy Mice in a Strain Dependent Manner  

PubMed Central

Many studies on probiotics are aimed at restoring immune homeostasis in patients to prevent disease recurrence or reduce immune-mediated pathology. Of equal interest is the use of probiotics in sub-clinical situations, which are characterized by reduced immune function or low-grade inflammation, with an increased risk of infection or disease as a consequence. Most mechanistic studies focus on the use of probiotics in experimental disease models, which may not be informative for these sub-clinical conditions. To gain better understanding of the effects in the healthy situation, we investigated the immunomodulatory effects of two Lactobacillus probiotic strains, i.e. L. plantarum WCFS1 and L. salivarius UCC118, and a non-probiotic lactococcus strain, i.e. L. lactis MG1363, in healthy mice. We studied the effect of these bacteria on the systemic adaptive immune system after 5 days of administration. Only L. plantarum induced an increase in regulatory CD103+ DC and regulatory T cell frequencies in the spleen. However, all three bacterial strains, including L. lactis, reduced specific splenic T helper cell cytokine responses after ex vivo restimulation. The effect on IFN-?, IL5, IL10, and IL17 production by CD4+ and CD8+ T cells was dependent on the strain administered. A shared observation was that all three bacterial strains reduced T helper 2 cell frequencies. We demonstrate that systemic immunomodulation is not only observed after treatment with probiotic organisms, but also after treatment with non-probiotic bacteria. Our data demonstrate that in healthy mice, lactobacilli can balance T cell immunity in favor of a more regulatory status, via both regulatory T cell dependent and independent mechanisms in a strain dependent manner. PMID:23056616

Smelt, Maaike J.; de Haan, Bart J.; Bron, Peter A.; van Swam, Iris; Meijerink, Marjolein; Wells, Jerry M.; Faas, Marijke M.; de Vos, Paul

2012-01-01

368

Physiology and Substrate Specificity of Two Closely Related Amino Acid Transporters, SerP1 and SerP2, of Lactococcus lactis.  

PubMed

The serP1 and serP2 genes found adjacently on the chromosome of Lactococcus lactis strains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transports l-serine, l-threonine, and l-cysteine with high affinity. Affinity constants (Km) are in the 20 to 40 ?M range. SerP2 is a dl-alanine/dl-serine/glycine transporter. The preferred substrate appears to be dl-alanine for which the affinities were found to be 38 and 20 ?M for the d and l isomers, respectively. The common substrate l-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 ?M, respectively. Growth experiments demonstrate that SerP1 is the main l-serine transporter responsible for optimal growth in media containing free amino acids as the sole source of amino acids. SerP2 is able to replace SerP1 in this role only in medium lacking the high-affinity substrates l-alanine and glycine. SerP2 plays an adverse role for the cell by being solely responsible for the uptake of toxic d-serine. The main function of SerP2 is in cell wall biosynthesis through the uptake of d-alanine, an essential precursor in peptidoglycan synthesis. SerP2 has overlapping substrate specificity and shares 42% sequence identity with CycA of Escherichia coli, a transporter whose involvement in peptidoglycan synthesis is well established. No evidence was obtained for a role of SerP1 and SerP2 in the excretion of excess amino acids during growth of L. lactis on protein/peptide-rich media. PMID:25535271

Noens, Elke E E; Lolkema, Juke S

2015-03-01

369

Combined transcriptome and proteome analysis of Bifidobacterium animalis subsp. lactis BB-12 grown on xylo-oligosaccharides and a model of their utilization.  

PubMed

Recent studies have demonstrated that xylo-oligosaccharides (XOS), which are classified as emerging prebiotics, selectively enhance the growth of bifidobacteria in general and of Bifidobacterium animalis subsp. lactis strains in particular. To elucidate the metabolism of XOS in the well-documented and widely used probiotic strain B. animalis subsp. lactis BB-12, a combined proteomic and transcriptomic approach was applied, involving DNA microarrays, real-time quantitative PCR (qPCR), and two-dimensional difference gel electrophoresis (2D-DIGE) analyses of samples obtained from cultures grown on either XOS or glucose. The analyses show that 9 of the 10 genes that encode proteins predicted to play a role in XOS catabolism (i.e., XOS-degrading and -metabolizing enzymes, transport proteins, and a regulatory protein) were induced by XOS at the transcriptional level, and the proteins encoded by three of these (?-d-xylosidase, sugar-binding protein, and xylose isomerase) showed higher abundance on XOS. Based on the obtained results, a model for the catabolism of XOS in BB-12 is suggested, according to which the strain utilizes an ABC (ATP-binding cassette) transport system (probably for oligosaccharides) to bind XOS on the cell surface and transport them into the cell. XOS are then degraded intracellularly through the action of xylanases and xylosidases to d-xylose, which is subsequently metabolized by the d-fructose-6-P shunt. The findings obtained in this study may have implications for the design of a synbiotic application containing BB-12 and the XOS used in the present study. PMID:20851982

Gilad, Ofir; Jacobsen, Susanne; Stuer-Lauridsen, Birgitte; Pedersen, Martin Bastian; Garrigues, Christel; Svensson, Birte

2010-11-01

370

Isolation of a bacteriocin-producing lactococcus lactis and application of its bacteriocin to manage spoilage bacteria in high-value marine fish under different storage temperatures.  

PubMed

The bacteriocins of lactic acid bacteria have considerable potential for biopreservation. The Lactococcus lactis strain PSY2 (GenBank account no. JF703669) isolated from the surface of marine perch Perca flavescens produced antibacterial activity against pathogenic and spoilage-causing Gram-positive and Gram-negative bacteria viz. Arthrobacter sp., Acinetobacter sp., Bacillus subtilis, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus and possessed broad inhibitory spectrum. The biopreservative efficacy of the bacteriocin PSY2 was evaluated using fillets of reef cod, Epinephelus diacanthus. The fillets (10g) were sprayed with 2.0ml of 1,600AU/ml bacteriocin, wrapped and kept under different storage temperatures viz., 4, 0 and -18C. The biopreservative extended the shelf-life of fillets stored at 4C to >21days as against <14days observed in the untreated samples. The total count of spoilage bacteria was reduced by 2.5 logarithmic units in the treated sample during the 14th day of storage as against the control. Chemical analysis revealed a significant change (P?lactis PSY2 gave increased protection against spoilage bacteria and offers an alternative for the preservation of high-value sea foods. PMID:22555500

Sarika, A R; Lipton, A P; Aishwarya, M S; Dhivya, R S

2012-07-01

371

Surface Proteome Analysis of a Natural Isolate of Lactococcus lactis Reveals the Presence of Pili Able to Bind Human Intestinal Epithelial Cells*  

PubMed Central

Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells. PMID:24002364

Meyrand, Mickael; Guillot, Alain; Goin, Mlodie; Furlan, Sylviane; Armalyte, Julija; Kulakauskas, Saulius; Cortes-Perez, Naima G.; Thomas, Ginette; Chat, Sophie; Pchoux, Christine; Dupres, Vincent; Hols, Pascal; Dufrne, Yves F.; Trugnan, Germain; Chapot-Chartier, Marie-Pierre

2013-01-01

372

Determination of the Domain of the Lactobacillus delbrueckii subsp. bulgaricus Cell Surface Proteinase PrtB Involved in Attachment to the Cell Wall after Heterologous Expression of the prtB Gene in Lactococcus lactis  

Microsoft Academic Search

Belonging to the subtilase family, the cell surface proteinase (CSP) PrtB of Lactobacillus delbrueckii subsp. bulgaricus differs from other CSPs synthesized by lactic acid bacteria. Expression of the prtB gene under its own promoter was shown to complement the proteinase-deficient strain MG1363 (PrtP PrtM )o fLactococcus lactis subsp. cremoris. Surprisingly, the maturation process of PrtB, unlike that of lactococcal CSP

Jacques-Edouard Germond; Michele Delley; Christophe Gilbert; Daniele Atlan

2003-01-01

373

Lait 84 (2004) 207214 INRA, EDP Sciences, 2003  

E-print Network

these goals, we characterized the -Gal structural gene of Lactobacillus plantarum ATCC8014. This gene, as well are specific to each application. Active -Gals could be produced in L. lactis and resulting strains ­ Vers des bactéries lactiques probiotiques pour l'élimination des sucres de type raffinose dans les

Boyer, Edmond

374

Variable Bacteriocin Production in the Commercial Starter Lactococcus lactis DPC4275 Is Linked to the Formation of the Cointegrate Plasmid pMRC02  

PubMed Central

Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications. PMID:14711623

Trotter, Maeve; McAuliffe, Olivia E.; Fitzgerald, Gerald F.; Hill, Colin; Ross, R. Paul; Coffey, Aidan

2004-01-01

375

Activation of mRNA translation by phage protein and low temperature: the case of Lactococcus lactis abortive infection system AbiD1  

PubMed Central

Background Abortive infection (Abi) mechanisms comprise numerous strategies developed by bacteria to avoid being killed by bacteriophage (phage). Escherichia coli Abis are considered as mediators of programmed cell death, which is induced by infecting phage. Abis were also proposed to be stress response elements, but no environmental activation signals have yet been identified. Abis are widespread in Lactococcus lactis, but regulation of their expression remains an open question. We previously showed that development of AbiD1 abortive infection against phage bIL66 depends on orf1, which is expressed in mid-infection. However, molecular basis for this activation remains unclear. Results In non-infected AbiD1+ cells, specific abiD1 mRNA is unstable and present in low amounts. It does not increase during abortive infection of sensitive phage. Protein synthesis directed by the abiD1 translation initiation region is also inefficient. The presence of the phage orf1 gene, but not its mutant AbiD1R allele, strongly increases abiD1 translation efficiency. Interestingly, cell growth at low temperature also activates translation of abiD1 mRNA and consequently the AbiD1 phenotype, and occurs independently of phage infection. There is no synergism between the two abiD1 inducers. Purified Orf1 protein binds mRNAs containing a secondary structure motif, identified within the translation initiation regions of abiD1, the mid-infection phage bIL66 M-operon, and the L. lactis osmC gene. Conclusion Expression of the abiD1 gene and consequently AbiD1 phenotype is specifically translationally activated by the phage Orf1 protein. The loss of ability to activate translation of abiD1 mRNA determines the molecular basis for phage resistance to AbiD1. We show for the first time that temperature downshift also activates abortive infection by activation of abiD1 mRNA translation. PMID:19173723

Bidnenko, Elena; Chopin, Alain; Ehrlich, S Dusko; Chopin, Marie-Christine

2009-01-01

376

Efficacy of the direct-fed microbial Enterococcus faecium alone or in combination with Saccharomyces cerevisiae or Lactococcus lactis during induced subacute ruminal acidosis.  

PubMed

This study aimed at investigating Enterococcus faecium alone or E. faecium in combination with Saccharomyces cerevisiae or Lactococcus lactis during a subacute ruminal acidosis (SARA) challenge. Four ruminally fistulated Holstein dairy cows were assigned to the following treatments in a 44 Latin square design: (1) control (CON); (2) E. faecium (EF); (3) EF + S. cerevisiae (EFSC); (4) EF + L. lactis DSM 11037 (EFLL). Each experimental period consisted of 18 d of adaptation to the respective direct-fed microbial, 3 d of SARA challenge, and 7d of rest. Rumen pH was recorded every 10 min over 24 h on d 17 of adaptation, d 2 of SARA, and d 6 of rest. On the last day of adaptation, SARA, and rest, samples of rumen content (0 and 3 h after feeding) were taken for volatile fatty acids, lactate, vitamin B12, rumen microbes, and lipopolysaccharides determination. Blood samples (0 and 6 h after feeding) were taken for the measurement of acute-phase proteins. Dry matter intake and milk yield were recorded daily. During SARA, mean rumen pH with EFSC (5.94) was not different from that of EFLL (5.95) and tended to be higher than with CON (5.82) or EF (5.82). Postfeeding vitamin B12 concentrations in the rumen were greater with EFSC (134.5ng/g) than with EF (99.6ng/g) and tended to be greater when compared with CON (101.2ng/g) or EFLL (104.9ng/g). During rest, prefeed vitamin B12 was greater with EFSC (166.5ng/g) compared with CON (132.3ng/g). The EFSC treatment did better than EF alone on pH characteristics during adaptation and SARA and on maintenance of ruminal vitamin B12 status during SARA. Milk yield drop from d 1 to 3 of SARA was smaller with EFSC (-0.8kg/d), EF (-0.9kg/d), or EFLL (-0.9kg/d) compared with CON (-7.5kg/d). PMID:25465534

Chiquette, J; Lagrost, J; Girard, C L; Talbot, G; Li, S; Plaizier, J C; Hindrichsen, I K

2015-01-01

377

Recombinant Kluyveromyces lactis expressing highly pathogenic porcine reproductive and respiratory syndrome virus GP5 elicits mucosal and cell-mediated immune responses in mice  

PubMed Central

Currently, killed-virus and modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are used to control porcine reproductive and respiratory syndrome. However, both types of vaccines have inherent drawbacks; accordingly, the development of novel PRRSV vaccines is urgently needed. Previous studies have suggested that yeast possesses adjuvant activities, and it has been used as an expression vehicle to elicit immune responses to foreign antigens. In this report, recombinant Kluyveromyces lactis expressing GP5 of HP-PRRSV (Yeast-GP5) was generated and immune responses to this construct were analyzed in mice. Intestinal mucosal PRRSV-specific sIgA antibody and higher levels of IFN-? in spleen CD4+ and CD8+ T cells were induced by oral administration of Yeast-GP5. Additionally, Yeast-GP5 administered subcutaneously evoked vigorous cell-mediated immunity, and PRRSV-specific lymphocyte proliferation and IFN-? secretion were detected in the splenocytes of mice. These results suggest that Yeast-GP5 has the potential for use as a vaccine for PRRSV in the future. PMID:24378591

Zhao, Haiyan; Wang, Yalan; Ma, Zhitao; Wang, Yongqiang

2014-01-01

378

Effects of ingesting milk fermented by Lactococcus lactis H61 on skin health in young women: a randomized double-blind study.  

PubMed

We conducted a randomized double-blind trial to evaluate the effects of fermented milk produced using only Lactococcus lactis strain H61 as a starter bacterium (H61-fermented milk) on the general health and various skin properties of young women. Healthy female volunteers (n=23; age=19-21r) received H61-fermented milk (10(10) cfu of strain H61/d) or conventional yogurt (10(10) cfu of both Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus per day), as a reference food, daily for 4 wk. Before and at the end of 4 wk, blood samples were taken, and skin hydration (inner forearms and cheek) and melanin content, elasticity, and sebum content (cheek only) were measured. Skin hydration at the inner forearm was higher at wk 4 than at wk 0 in both groups. Sebum content in cheek rose significantly after intervention in the H61-fermented milk group, but not the conventional yogurt group. Other skin parameters did not differ in either group. Serum analysis showed that total protein concentration and platelet count were elevated and reactive oxygen species decreased in both groups after the intervention. Although H61-fermented milk and conventional yogurt had similar effects on skin status and some blood characteristics of participants, an increase of sebum content in cheek is preferable to H61-fermented milk. As skin lipids contribute to maintaining the skin barrier, H61-fermented milk would provide beneficial effects on skin for young women. PMID:25022690

Kimoto-Nira, H; Nagakura, Y; Kodama, C; Shimizu, T; Okuta, M; Sasaki, K; Koikawa, N; Sakuraba, K; Suzuki, C; Suzuki, Y

2014-09-01

379

The C-terminus of nisin is important for the ABC transporter NisFEG to confer immunity in Lactococcus lactis.  

PubMed

The lantibiotic nisin is a small 3.4kDa antimicrobial peptide, which acts against Gram-positive bacteria in the nmol/L range. Nisin is produced and secreted by several Lactococcus lactis strains to ensure advantages against other bacteria in their habitat. Nisin contains five specific lanthionine rings of which the first two are important for Lipid II binding and the last two are crucial for the pore formation in the membrane. To gain immunity against nisin, the producing strain is expressing an ABC transporter called NisFEG, which expels nisin from the membrane. As a result six to eightfold more nisin is needed to affect the cells. The hydrolysis of ATP by NisFEG is required for this immunity as shown by a mutant, where the ATP hydrolysis is disrupted (NisFH181A EG). Furthermore, NisFEG recognizes the C-terminus of nisin, since deletion of the last six amino acids as well as of the last ring lowered the fold of immunity displayed by NisFEG. PMID:25176038

AlKhatib, Zainab; Lagedroste, Marcel; Zaschke, Julia; Wagner, Manuel; Abts, Andr; Fey, Iris; Kleinschrodt, Diana; Smits, Sander H J

2014-10-01

380

Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis?  

PubMed Central

In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1? strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield. PMID:17085636

Saliola, Michele; Scappucci, Gina; De Maria, Ilaria; Lodi, Tiziana; Mancini, Patrizia; Falcone, Claudio

2007-01-01

381

Prevention of C-terminal autoprocessing of Lactococcus lactis SK11 cell-envelope proteinase by engineering of an essential surface loop.  

PubMed Central

The catalytic domain of the cell-envelope proteinase from Lactococcus lactis SK11 has various inserts, situated in external loops of the catalytic domain, compared with the related subtilisins. Protein engineering was employed to analyse the necessity and function of one of these extra loops (residues 205-219), that is predicted to be located in close proximity to the substrate-binding region and is susceptible to autoproteolysis. We constructed a deletion mutant which lacks 14 residues of this surface loop and subsequently introduced various insertion cassettes coding either for the original loop with three mutations (E205S/E218T/M219S: triple-mutant proteinase) or for neutral spacers (1, 4, 7 and 16 serine residues). Engineered proteinases were analysed for activity, (auto)processing, and cleavage specificity. The presence of residues 205-219 is shown to be essential for proteolytic activity, as only triple-mutant proteinase retained activity towards casein substrates. The triple-mutant proteinase was found to be defective in C-terminal autoprocessing, and subsequent release from the lactococcal cell envelope in a calcium-free medium, indicative of either an altered proteolytic specificity or altered accessibility of the processing site. The specificity change appears to be subtle, as only small differences were found between wild-type and triple-mutant proteinase in the breakdown of casein substrates. Images Figure 4 Figure 6 PMID:7945226

Bruinenberg, P G; de Vos, W M; Siezen, R J

1994-01-01

382

Comparison of killing of gram-negative and gram-positive bacteria by pure singlet oxygen. [Salmonella typhimurium; Escherichia coli; Sarcina lutea; Staphylococcus aureus; Streptococcus lactis; Streptococcus faecalis  

SciTech Connect

Gram-negative and gram-positive bacteria were found to display different sensitivities to pure singlet oxygen generated outside of cells. Killing curves for Salmonella typhimurium and Escherichia coli strains were indicative of multihit killing, whereas curves for Sarcina lutea, Staphylococcus aureus, Streptococcus lactis, and Streptococcus faecalis exhibited single-hit kinetics. The S. typhimurium deep rough strain TA1975, which lacks nearly all of the cell wall lipopolysaccharide coat and manifests concomitant enhancement of penetration by some exogenous substances, responded to singlet oxygen with initially faster inactivation than did the S. typhimurium wild-type strain, although the maximum rates of killing appeared to be quite similar. The structure of the cell wall thus plays an important role in susceptibility to singlet oxygen. The outer membrane-lipopolysaccharide portion of the gram-negative cell wall initially protects the bacteria from extracellular singlet oxygen, although it may also serve as a source for secondary reaction products which accentuate the rates of cell killing. S. typhimurium and E. coli strains lacking the cellular antioxidant, glutathione, showed no difference from strains containing glutathione in response to the toxic effects of singlet oxygen. Strains of Sarcina lutea and Staphylococcus aureus that contained carotenoids, however, were far more resistant to singlet oxygen lethality than were both carotenoidless mutants of the same species and other gram-positive species lacking high levels of protective carotenoids.

Dahl, T.A.; Midden, W.R. (Bowling Green State Univ., OH (USA)); Hartman, P.E. (Johns Hopkins Univ., Baltimore, MD (USA))

1989-04-01

383

Enhanced free fatty acid production by codon-optimized Lactococcus lactis acyl-ACP thioesterase gene expression in Escherichia coli using crude glycerol.  

PubMed

Fatty acid production and composition are determined by the type of acyl-acyl carrier protein thioesterases (acyl-ACP TEs) expressed in Escherichia coli. Bacterial acyl-ACP TEs from Lactococcus lactis (SGJS47), Enterococcus faecalis (SGJS49), and Burkholderia cepacia (SGJS50) were codon-optimized and expressed in E. coli for enhanced fatty acid production. Samples were extracted at the lag, log, and stationary phases of cell growth, and gene expression levels of the codon optimized acy-ACP TEs as well as fatty acid production were monitored. At 24h after initiation of gene expression, the OPLlTE expression level and fatty acid production in SGJS47 increased up to 15.8-fold and 3.2-fold compared to the control and other recombinant strains, respectively. Additionally, in SGJS47, improvement in free fatty acid (FFA) composition, high-specificity production of short-chain fatty acids (C8, C10) and unsaturated fatty acids (C16:1) was achieved in crude glycerol medium condition. Compared with control strain, the percentage of FFAs (C8 and C10) was enhanced by approximately 16- to 21-fold, C16:1 FFA ratio increased approximately 18-fold. Observation of codon-optimized acyl-ACP TE genes expression level in E. coli may be useful for understanding mechanisms towards improving fatty acid production. Engineered strains have the potential to overproduce specific FFAs and thereby reduce the cost of fatty acid production by using industrially inexpensive carbon sources. PMID:25442943

Lee, Sunhee; Park, Soohyun; Park, Chulhwan; Pack, Seung Pil; Lee, Jinwon

2014-12-01

384

Maintenance of Very Long Telomeres by Recombination in the Kluyveromyces lactis stn1-M1 Mutant Involves Extreme Telomeric Turnover, Telomeric Circles, and Concerted Telomeric Amplification  

PubMed Central

Some cancers utilize the recombination-dependent process of alternative lengthening of telomeres (ALT) to maintain long heterogeneous telomeres. Here, we studied the recombinational telomere elongation (RTE) of the Kluyveromyces lactis stn1-M1 mutant. We found that the total amount of the abundant telomeric DNA in stn1-M1 cells is subject to rapid variation and that it is likely to be primarily extrachromosomal. Rad50 and Rad51, known to be required for different RTE pathways in Saccharomyces cerevisiae, were not essential for the production of either long telomeres or telomeric circles in stn1-M1 cells. Circles of DNA containing telomeric repeats (t-circles) either present at the point of establishment of long telomeres or introduced later into stn1-M1 cells each led to the formation of long tandem arrays of the t-circle's sequence, which were incorporated at multiple telomeres. These tandem arrays were extraordinarily unstable and showed evidence of repeated rounds of concerted amplification. Our results suggest that the maintenance of telomeres in the stn1-M1 mutant involves extreme turnover of telomeric sequences from processes including both large deletions and the copying of t-circles. PMID:22645309

Xu, Jianing

2012-01-01

385

Restriction and modification activities from Streptococcus lactis ME2 are encoded by a self-transmissible plasmid, pTN20, that forms cointegrates during mobilization of lactose-fermenting ability.  

PubMed Central

A self-transmissible (Tra+) plasmid encoding determinants for restriction and modification activities (R+/M+) from Streptococcus lactis ME2 was isolated and characterized. The 28-kilobase (kb) plasmid (pTN20) was detected in lactose-fermenting (Lac+) transconjugants generated from matings between S. lactis N1, and ME2 variant, and a plasmid-free recipient, S. lactis LM2301. The plaquing efficiencies of prolate- and small isometric-headed phages were reduced on transconjugants containing either pTN20 (R+/M+ Tra+) or 100-kb plasmids encoding Lac+, R+/M+, and Tra+. Lac+ transconjugants which harbored pTR1040 (Lac+) and pTN20 (R+/M+) were phenotypically R-/M- and transferred Lac+ at low frequency in subsequent matings to give rise to 100-kb R+/M+ plasmids. R+/M+ activities and high-frequency conjugal transfer ability were detected in Lac+ transconjugants that contained pTR1041 (Lac+) and pTN20 (R+/M+). No 100-kb R+/M+ plasmids were recovered after these matings, suggesting that pTR1041 was mobilized by pTN20 through a process that resembled plasmid donation. pTR1041 was identical to pTR1040 but contained an additional 3.3-kb DNA fragment. These data suggested that phenotypic expression of R+/M+ and Tra+ is affected by coresident Lac+ plasmids. Restriction enzyme analysis and hybridization reactions demonstrated that the 100-kb R+/M+ plasmid was formed by a cointegration event between pTR1040 (Lac+) and pTN20 (R+/M+ Tra+) during conjugal transfer via a conductive-type process. This is the first report that defines self-transmissible restriction and modification plasmids in the lactic streptococci. Images PMID:2841286

Higgins, D L; Sanozky-Dawes, R B; Klaenhammer, T R

1988-01-01

386

Oxalate-Degrading Activity in Bifidobacterium animalis subsp. lactis: Impact of Acidic Conditions on the Transcriptional Levels of the Oxalyl Coenzyme A (CoA) Decarboxylase and Formyl-CoA Transferase Genes ?  

PubMed Central

Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyperabsorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and can be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis and reducing the risk of kidney stone development. In this study, the oxalate-degrading activities of 14 bifidobacterial strains were measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-coenzyme A (CoA) decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of Bifidobacterium animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unraveling the presence of two other independently transcribed open reading frames, potentially responsible for the ability of B. animalis subsp. lactis to degrade oxalate. pH-controlled batch fermentations revealed that acidic conditions were a prerequisite for a significant oxalate degradation rate, which dramatically increased in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Oxalate-preadapted cells also showed a strong induction of the genes potentially involved in oxalate catabolism, as demonstrated by a transcriptional analysis using quantitative real-time reverse transcription-PCR. These findings provide new insights into the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease. PMID:20601517

Turroni, Silvia; Bendazzoli, Claudia; Dipalo, Samuele C. F.; Candela, Marco; Vitali, Beatrice; Gotti, Roberto; Brigidi, Patrizia

2010-01-01

387

Modelling the combined effects of structured food model system and lactic acid on Listeria innocua and Lactococcus lactis growth in mono- and coculture.  

PubMed

A new class of predictive model developed in a liquid system is extended in order to quantify gelatin gel matrix structure effects on the growth of Listeria innocua and Lactococcus lactis (both in mono- and coculture, and both producing mainly lactic acid). It was observed that gelatin does not only act as a structuring agent but also alters the buffering capacity of the medium. Model extension occurs in two stages, describing chemical and microbiological processes, respectively. Firstly, equations relating undissociated lactic acid concentration and total lactic acid concentration on the one hand, and undissociated lactic acid concentration and pH on the other hand, are extended to account for the effects of gelatin concentration. Secondly, these equations are incorporated into the growth model to describe the combined effect of gelatin concentration, (undissociated) lactic acid and pH on the growth of either microorganism. The description of the model is in good agreement with the experimental data acquired in monoculture conditions. In a subsequent model validation step, when gelatin concentration and total lactic acid profile of the coculture experiments are used as inputs, the developed growth model consisting of condensed knowledge extracted from the monoculture experiments, is able to predict accurately the interaction effect occurring in coculture. The study suggests that, on the one hand, the extent of the effects of undissociated lactic acid and pH on microbial growth in structured food systems can be modified by the increase in buffering capacity, which can protect microorganisms and eventually promote higher levels of cell growth in comparison with liquid culture conditions. On the other hand, food matrix structure, in casu the gelatin, reduces the rate of microbial multiplication. Both effects are incorporated in the growth model developed in this research. PMID:17629978

Antwi, M; Bernaerts, K; Van Impe, J F; Geeraerd, A H

2007-11-30

388

Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 ?-d-xylosidase/?-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12  

PubMed Central

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a ?-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-?-d-xylopyranoside (pNPX), para-nitrophenyl-?-L-arabinofuranoside (pNPA), ?-(1???4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 24, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I???V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for pNPA:pNPX of 1:25. BXA43 was stable below 40C and at pH4.08.0 and showed maximum activity at pH5.5 and 50C. Km and kcat for pNPX were 15.6??4.2mM and 60.6??10.8s-1, respectively, and substrate inhibition became apparent above 18mM pNPX. Similar kinetic parameters and catalytic efficiency values were reported for ?-d-xylosidase (XynB3) from Geobacillus stearothermophilus T?6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to pNPX. BXA43 showed clearly metal inhibition for Zn2+ and Ag+, which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg505Tyr506 present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus. PMID:24025736

2013-01-01

389

Administration of a probiotic associated with nasal vaccination with inactivated Lactococcus lactis-PppA induces effective protection against pneumoccocal infection in young mice.  

PubMed

Streptococcus pneumoniae is a serious public health problem, especially in developing countries, where available vaccines are not part of the vaccination calendar. We evaluated different respiratory mucosa immunization protocols that included the nasal administration of Lactococcus lactis-pneumococcal protective protein A (PppA) live, inactivated, and in association with a probiotic (Lc) to young mice. The animals that received Lc by the oral and nasal route presented the highest levels of immunoglobulin (Ig)A and IgG anti-PppA antibodies in bronchoalveolar lavages (BAL) and IgG in serum, which no doubt contributed to the protection against infection. However, only the groups that received the live and inactivated vaccine associated with the oral administration of the probiotic were able to prevent lung colonization by S. pneumoniae serotypes 3 and 14 in a respiratory infection model. This would be related to a preferential stimulation of the T helper type 1 (Th1) cells at local and systemic levels and with a moderate Th2 and Th17 response, shown by the cytokine profile induced in BAL and by the results of the IgG1/IgG2a ratio at local and systemic levels. Nasal immunization with the inactivated recombinant strain associated with oral Lc administration was able to stimulate the specific cellular and humoral immune response and afford protection against the challenge with the two S. pneumoniae serotypes. The results obtained show the probiotic-inactivated vaccine association as a valuable alternative for application to human health, especially in at-risk populations, and are the first report of a safe and effective immunization strategy using an inactivated recombinant strain. PMID:20002449

Vintii, E; Villena, J; Alvarez, S; Medina, M

2010-03-01

390

Safety assessment of potential probiotic lactic acid bacterial strains Lactobacillus rhamnosus HN001, Lb. acidophilus HN017, and Bifidobacterium lactis HN019 in BALB/c mice.  

PubMed

The general safety of immune-enhancing lactic acid bacteria (LAB) strains Lactobacillus rhamnosus HN001 (DR20), Lb. acidophilus HN017, and Bifidobacterium lactis HN019 (DR10) was investigated in a feeding trial. Groups of BALB/c mice were orally administered test LAB strains or the commercial reference strain Lb. acidophilus LA-1 at 2.5 x 10(9), 5 x 10(10) or 2.5 x 10(12) colony forming units (CFU)/kg body weight/day for 4 weeks. Throughout this time, their feed intake, water intake, and live body weight were monitored. At the end of the 4 week observation period, samples of blood, liver, spleen, kidney, mesenteric lymph nodes, and gut tissues (ileum, caecum, and colon) were collected to determine: haematological parameters (red blood cell and platelet counts, haemoglobin concentration, mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration); differential leukocyte counts; blood biochemistry (plasma total protein, albumin, cholesterol, and glucose); mucosal histology (epithelial cell height, mucosal thickness, and villus height); and bacterial translocation to extra-gut tissues (blood, liver, spleen, kidney and mesenteric lymph nodes). DNA finger printing techniques were used to identify any viable bacterial strains recovered from these tissues. The results demonstrated that 4 weeks consumption of these LAB strains had no adverse effects on animals' general health status, haematology, blood biochemistry, gut mucosal histology parameters, or the incidence of bacterial translocation. A few viable LAB cells were recovered from the tissues of animals in both control and test groups, but DNA fingerprinting did not identify any of these as the inoculated strains. The results obtained in this study suggest that the potentially probiotic LAB strains HN001, HN017, and HN019 are non-toxic for mice and are therefore likely to be safe for human use. PMID:10857928

Zhou, J S; Shu, Q; Rutherfurd, K J; Prasad, J; Birtles, M J; Gopal, P K; Gill, H S

2000-05-25

391

Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 ?-d-xylosidase/?-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12.  

PubMed

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a ?-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-?-d-xylopyranoside (pNPX), para-nitrophenyl-?-L-arabinofuranoside (pNPA), ?-(1???4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2-4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I?-?V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for pNPA:pNPX of 1:25. BXA43 was stable below 40C and at pH4.0-8.0 and showed maximum activity at pH5.5 and 50C. Km and kcat for pNPX were 15.6??4.2mM and 60.6??10.8s-1, respectively, and substrate inhibition became apparent above 18mM pNPX. Similar kinetic parameters and catalytic efficiency values were reported for ?-d-xylosidase (XynB3) from Geobacillus stearothermophilus T?6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to pNPX. BXA43 showed clearly metal inhibition for Zn2+ and Ag+, which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg505Tyr506 present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus. PMID:24025736

Viborg, Alexander Holm; Srensen, Kim Ib; Gilad, Ofir; Steen-Jensen, Daniel Bisgaard; Dilokpimol, Adiphol; Jacobsen, Susanne; Svensson, Birte

2013-01-01

392

The d-2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis?  

PubMed Central

Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branched-chain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding l-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (Vmax/Km ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted d-2-hydroxyacids but not l-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the d-mandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of d-2-hydroxyacid dehydrogenases which is unrelated to the well-described d-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD+ necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds. PMID:19047348

Chambellon, Emilie; Rijnen, Liesbeth; Lorquet, Frdrique; Gitton, Christophe; van Hylckama Vlieg, Johan E. T.; Wouters, Jeroen A.; Yvon, Mireille

2009-01-01

393

Three Target Genes for the Transcriptional Activator Cat8p of Kluyveromyces lactis: Acetyl Coenzyme A Synthetase Genes KlACS1 and KlACS2 and Lactate Permease Gene KlJEN1  

PubMed Central

The aerobic yeast Kluyveromyces lactis and the predominantly fermentative Saccharomyces cerevisiae share many of the genes encoding the enzymes of carbon and energy metabolism. The physiological features that distinguish the two yeasts appear to result essentially from different organization of regulatory circuits, in particular glucose repression and gluconeogenesis. We have isolated the KlCAT8 gene (a homologue of S. cerevisiae CAT8, encoding a DNA binding protein) as a multicopy suppressor of a fog1 mutation. The Fog1 protein is a homologue of the Snf1 complex components Gal83p, Sip1p, and Sip2p of S. cerevisiae. While CAT8 controls the key enzymes of gluconeogenesis in S. cerevisiae, KlCAT8 of K. lactis does not (I. Georis, J. J. Krijger, K. D. Breunig, and J. Vandenhaute, Mol. Gen. Genet. 264:193203, 2000). We therefore examined possible targets of KlCat8p. We found that the acetyl coenzyme A synthetase genes, KlACS1 and KlACS2, were specifically regulated by KlCAT8, but very differently from the S. cerevisiae counterparts. KlACS1 was induced by acetate and lactate, while KlACS2 was induced by ethanol, both under the control of KlCAT8. Also, KlJEN1, encoding the lactate-inducible and glucose-repressible lactate permease, was found under a tight control of KlCAT8. PMID:11514507

Lodi, Tiziana; Saliola, Michele; Donnini, Claudia; Goffrini, Paola

2001-01-01

394

Abstract We describe here aspects of the anatomy of two "Epulopiscium" morphotypes, unusually large bacte-  

E-print Network

of herbivorous surgeonfish (Acanthuri- dae), were collected around the Great Barrier Reef of Australia, preserved in surgeonfish from Australia's Great Barrier Reef (Clements et al. 1989). Electron microscopy of sections

Pawlowski, Wojtek

395

Hydrogen-driven asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol by Ralstonia eutropha transformant expressing alcohol dehydrogenase from Kluyveromyces lactis  

PubMed Central

Background Conversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alcohol dehydrogenase from Kluyveromyces lactis in a hydrogen-oxidizing bacterium: Ralstonia eutropha. This is the first report of hydrogen-driven in vivo coupling reaction of the alcohol dehydrogenase and indigenous soluble NAD-reducing hydrogenase. Asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol, which is a commercial building block for antibacterial agents, was performed using the transformant as the microbial cell catalyst. Results The two enzymes coupled in vitro in vials without a marked decrease of reactivity during the 20 hr reaction because of the hydrogenase reaction, which generates no by-product that affects enzymes. Alcohol dehydrogenase was expressed functionally in R. eutropha in an activity level equivalent to that of indigenous NAD-reducing hydrogenase under the hydrogenase promoter. The hydrogen-driven in vivo coupling reaction proceeded only by the transformant cell without exogenous addition of a cofactor. The decrease of reaction velocity at higher concentration of hydroxyacetone was markedly reduced by application of an in vivo coupling system. Production of (R)-1,2-propanediol (99.8% e.e.) reached 67.7 g/l in 76 hr with almost a constant rate using a jar fermenter. The reaction velocity under 10% PH2 was almost equivalent to that under 100% hydrogen, indicating the availability of crude hydrogen gas from various sources. The in vivo coupling system enabled cell-recycling as catalysts. Conclusions Asymmetric reduction of hydroxyacetone by a coupling reaction of the two enzymes continued in both in vitro and in vivo systems in the presence of hydrogen. The in vivo reaction system using R. eutropha transformant expressing heterologous alcohol dehydrogenase showed advantages for practical usage relative to the in vitro coupling system. The results suggest a hopeful perspective of the hydrogen-driven bioprocess as an environmentally outstanding method to achieve industrial green innovation. Hydrogen-oxidizing bacteria can be useful hosts for the development of hydrogen-driven microbial cell factories. PMID:23305396

2013-01-01

396

Phenotypical analysis of the Lactobacillus rhamnosus GG fimbrial spaFED operon: surface expression and functional characterization of recombinant SpaFED pili in Lactococcus lactis.  

PubMed

A noticeable genomic feature of many piliated Gram-positive bacterial species is the presence of more than one pilus-encoding operon. Paradigmatically, the gut-adapted Lactobacillus rhamnosus GG strain contains two different fimbrial operons in its genome. However, whereas one of these operons (called spaCBA) is encoding for the functionally mucus-/collagen-binding SpaCBA pilus, for the other operon (called spaFED) any native expression of the SpaFED-called pili is still the subject of some uncertainty. Irrespective of such considerations, we decided it would be of relevance or interest to decipher the gross structure of this pilus type, and as well assess its functional capabilities for cellular adhesion and immunostimulation. For this, and by following the approach we had used previously to explicate the immuno-properties of SpaCBA pili, we constructed nisin-inducible expression clones producing either wild-type or SpaF pilin-deleted surface-assembled L. rhamnosus GG SpaFED pili on Lactococcus lactis cells. Using these piliated lactococcal constructs, we found that the pilin-polymerized architecture of a recombinant-produced SpaFED pilus coincides with sequence-based functional predictions of the related pilins, and in fact is prototypical of those other sortase-dependent pilus-like structures thus far characterized for piliated Gram-positive bacteria. Moreover, we confirmed that among the different pilin subunits encompassing spaFED operon-encoded pili, the SpaF pilin is a main adhesion determinant, and when present in the assembled structure can mediate pilus binding to mucus, certain extracellular matrix proteins, and different gut epithelial cell lines. However, somewhat unexpectedly, when recombinant SpaFED pili are surface-attached, we found that they could not potentiate the existing lactococcal cell-induced immune responses so elicited from intestinal- and immune-related cells, but rather instead, they could dampen them. Accordingly, we have now provided the first phenotypical description of a spaFED pilus operon, and with that furthered the functional understanding of surface piliation for a particular gut-commensalic genre of piliated Gram-positive bacteria. PMID:25415357

Rintahaka, Johanna; Yu, Xia; Kant, Ravi; Palva, Airi; von Ossowski, Ingemar

2014-01-01

397

Inhibition of the phosphoenolpyruvate:lactose phosphotransferase system and activation of a cytoplasmic sugar-phosphate phosphatase in Lactococcus lactis by ATP-dependent metabolite-activated phosphorylation of serine 46 in the phosphocarrier protein HPr.  

PubMed

Lactococcus lactis takes up lactose and the nonmetabolizable lactose analogue, thiomethyl-beta-galactoside (TMG), via the phosphoenolpyruvate:sugar phosphotransferase system (PTS) which couples sugar transport to sugar phosphorylation. Earlier studies had shown that TMG-phosphate, previously accumulated in L. lactis cells, is rapidly dephosphorylated in the cytoplasm and effluxes from the cells upon addition of glucose and that glucose inhibits further uptake of TMG. We have developed a vesicular system to analyze this regulatory mechanism and have used electroporation to shock proteins and membrane-impermeable metabolites into the vesicles. Uptake of TMG was dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate at low concentrations or extravesicular phosphoenolpyruvate at high concentrations. TMG uptake into osmotically shocked vesicles was only weakly inhibited, and expulsion of preaccumulated TMG was only slightly stimulated upon addition of glucose. Intravesicular (but not extravesicular) wild-type HPr of Bacillus subtilis completely restored the regulatory behavior observed in vivo when glucose was present in the external medium. Glucose could be replaced by intravesicular (but not extravesicular) fructose 1,6-diphosphate, gluconate 6-phosphate, or 2-phosphoglycerate, but not by other phosphorylated metabolites, in agreement with the allosteric activating effects of these compounds on HPr(Ser) kinase measured in vitro. Intravesicular mutant HPr(S46A) protein could not promote regulation of lactose permease activity when electroporated into the vesicles regardless of the presence or absence of glucose or the various phosphorylated metabolites, but the HPr(S46D) mutant protein promoted regulation, even in the absence of glucose or a metabolite, and HPr(H15A) was more effective than the wild-type protein in promoting regulation. Intravesicular wild-type and H15A HPrs, but not the S46A or S46D mutant proteins, were found to be phosphorylated by ATP under the conditions which promoted TMG efflux. In toluenized vesicles, the conditions which promoted TMG efflux also promoted TMG-P hydrolysis. These results establish for the first time that HPr serine phosphorylation by the ATP-dependent metabolite-activated HPr kinase regulates the expulsion of intracellular sugar-phosphate as well as the uptake of sugar via the PTS in L. lactis. PMID:8163482

Ye, J J; Reizer, J; Cui, X; Saier, M H

1994-04-22

398

Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 induce different age-related metabolic profiles revealed by 1H-NMR spectroscopy in urine and feces of mice.  

PubMed

Age-related dysbioses of intestinal microbiota and decline in the overall metabolic homeostasis are frequently found in the elderly. Probiotic supplementation may represent a way to prevent or reduce the senescence-associated metabolic disorders. The present study evaluated the metabolic impact of Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 supplementation in relation to age by analyzing urine and feces metabolic profiles using (1)H-nuclear magnetic resonance spectroscopy and multivariate analysis. Adult (3 mo old) and aged (16 mo old) mice received an oral supplementation of the 2 probiotics (1 10(9) colony-forming units/d each) or phosphate buffered saline (control) daily for 30 d. Urine and feces were collected for 48 h before the end of the study. Partial least squares-discriminant analysis showed that the urinary discriminant metabolites for the probiotic treatment included higher dimethylglycine in adult and aged mice, lower sarcosine and nicotinate in adult mice, higher N-methylnicotinamide in adult mice and lower N-methylnicotinamide in aged mice compared with their controls. These results indicate a probiotic-induced modulation of homocysteine and NAD metabolism pathways, which have important implications because these pathways are involved in essential cellular processes that can be altered in senescence. The probiotic supplementation also modified the fecal metabolic profiles, inducing in both adult and aged mice higher 4-hydroxyphenylacetate and lower xylose in treated mice compared with their control mice, whereas valerate was greater in treated adult mice and lower in treated aged mice compared with their controls. The ANOVA simultaneous component analysis on urinary and fecal metabolic profiling showed an age treatment interaction (P < 0.05), confirming the age-related modulation of the metabolic response to probiotic supplementation. The results suggest that L. acidophilus and B. lactis may prevent or reduce age-related metabolic dysfunction. PMID:23946343

Brasili, Elisa; Mengheri, Elena; Tomassini, Alberta; Capuani, Giorgio; Roselli, Marianna; Finamore, Alberto; Sciubba, Fabio; Marini, Federico; Miccheli, Alfredo

2013-10-01

399

De novo biosynthesis of linoleic acid and its conversion to the hydrocarbon (Z,Z)-6,9-heptadecadiene in the astigmatid mite, Carpoglyphus lactis: incorporation experiments with 13C-labeled glucose.  

PubMed

De novo biosynthesis of linoleic acid (LA) and its conversion to (Z,Z)-6,9-heptadecadiene were examined in Carpoglyphus lactis (Acarina, Carpoglyphidae). Experiments involving (13)C-administration using [1-(13)C]-d-glucose revealed that (13)C atoms were incorporated into LA of total lipid extracted from the mite, resulting in labeling of all even-numbered carbons. This result demonstrated that LA was produced from (13)C-labeled acetyl-CoA, which is indicative of direct de novo biosynthesis. In these feeding experiments involving [1-(13)C]-D-glucose, (13)C atoms were also incorporated into (Z,Z)-6,9-heptadecadiene, which is one of the major secretory components in the mite. The labeling pattern of (Z,Z)-6,9-heptadecadiene at odd-numbered carbons agreed well with that of LA after loss of the carboxyl carbon. It was concluded that the mites could stably convert LA into (Z,Z)-6,9-heptadecadiene without the dietary requirement of this essential fatty acid. PMID:24333472

Shimizu, Nobuhiro; Naito, Michiya; Mori, Naoki; Kuwahara, Yasumasa

2014-02-01

400

Geoffrey Layton Slack OBE (Mil), CBE, TD, BDS DDS, FDSRCS, FDS Glas, FFDRCSI, Dip Bact (1912-1991).  

PubMed

It is with some pride that the author worked in Geoffrey Slack's department from 1963 to 1967 and even retained a working relationship with him after that time. Slack was Professor of Dental Surgery (1959-1976) and later Professor of Community Dental Health (1976-1977) at The London Hospital Medical College, within the University of London. The change in titles came about as a result of recognition of his contribution to developments in public health and community dental care and services, for many of which he was directly responsible. He was Dental Dean from 1965 until 1969. Upon retirement in 1977 he became Emeritus Professor. In addition, he was Dean of the Faculty of Dental Surgery at the Royal College of Surgeons of England from 1974 to 1977. PMID:24585843

Gelbier, Stanley

2014-02-01