Loss of the intrinsic plasmid-encoded eps genes in Lactococcus lactis subsp. cremoris FC grown at elevated temperature abolishes exopolysaccharide biosynthesis

2021 ◽  
Vol 27 (2) ◽  
pp. 241-248
Author(s):  
Yayoi Gotoh ◽  
Toshinari Maruo ◽  
Kosei Tanaka ◽  
Satoshi Ohashi ◽  
Ken-ichi Yoshida ◽  
...  
Keyword(s):  
Elevated Temperature ◽  
Lactococcus Lactis ◽  
Exopolysaccharide Biosynthesis

scholarly journals Functional Analysis of the Lactococcus lactis galU and galE Genes and Their Impact on Sugar Nucleotide and Exopolysaccharide Biosynthesis

2001 ◽  
Vol 67 (7) ◽  
pp. 3033-3040 ◽  
Author(s):  
Ingeborg C. Boels ◽  
Ana Ramos ◽  
Michiel Kleerebezem ◽  
Willem M. de Vos
Keyword(s):  
Lactococcus Lactis ◽  
Sole Carbon Source ◽  
Northern Blot Analysis ◽  
Fold Increase ◽  
Activity Level ◽  
Wild Type ◽  
Degenerate Primers ◽  
Poor Growth ◽  
Wild Type Level ◽  
Exopolysaccharide Biosynthesis

ABSTRACT We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in L. lactis. The lactococcal galU gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The L. lactis galU gene could complement an Escherichia coli galU mutant, and overexpression of this gene in L. lactis under control of the inducible nisA promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDP-glucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the galE gene resulted in poor growth, undetectable intracellular levels of UDP-galactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the galE disruption mutant, while the level of EPS production was approximately one-half the wild-type level.

scholarly journals Molecular characterization of the plasmid‐encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis

1997 ◽  
Vol 24 (2) ◽  
pp. 387-397 ◽  
Author(s):  
Richard van Kranenburg ◽  
Joey D. Marugg ◽  
Iris I. Van Swam ◽  
Norwin J. Willem ◽  
Willem M. De Vos
Keyword(s):  
Gene Cluster ◽  
Molecular Characterization ◽  
Lactococcus Lactis ◽  
Exopolysaccharide Biosynthesis

scholarly journals Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

2001 ◽  
Vol 67 (1) ◽  
pp. 33-41 ◽  
Author(s):  
Ana Ramos ◽  
Ingeborg C. Boels ◽  
Willem M. de Vos ◽  
Helena Santos
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Pentose Phosphate ◽  
Biosynthetic Pathways ◽  
Glycolytic Intermediates ◽  
Phosphoglyceric Acid ◽  
Exopolysaccharide Biosynthesis ◽  
Fructose 1,6 Bisphosphate ◽  
Sugar Derivatives

ABSTRACT The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation.

scholarly journals Identification and Molecular Characterization of the Chromosomal Exopolysaccharide Biosynthesis Gene Cluster from Lactococcus lactis subsp. cremoris SMQ-461

2005 ◽  
Vol 71 (11) ◽  
pp. 7414-7425 ◽  
Author(s):  
N. Dabour ◽  
G. LaPointe
Keyword(s):  
Gene Cluster ◽  
Lactococcus Lactis ◽  
Repeat Unit ◽  
Raw Milk ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Biosynthesis Gene Cluster ◽  
Glycosyltransferase Gene ◽  
Reverse Transcription Pcr ◽  
Exopolysaccharide Biosynthesis

ABSTRACT The exopolysaccharide (EPS) capsule-forming strain SMQ-461 of Lactococcus lactis subsp. cremoris, isolated from raw milk, produces EPS with an apparent molecular mass of >1.6 × 106 Da. The EPS biosynthetic genes are located on the chromosome in a 13.2-kb region consisting of 15 open reading frames. This region is flanked by three IS1077-related tnp genes (L. lactis) at the 5′ end and orfY, along with an IS981-related tnp gene, at the 3′ end. The eps genes are organized in specific regions involved in regulation, chain length determination, biosynthesis of the repeat unit, polymerization, and export. Three (epsGIK) of the six predicted glycosyltransferase gene products showed low amino acid similarity with known glycosyltransferases. The structure of the repeat unit could thus be different from those known to date for Lactococcus. Reverse transcription-PCR analysis revealed that the eps locus is transcribed as a single mRNA. The function of the eps gene cluster was confirmed by disrupting the priming glycosyltransferase gene (epsD) in Lactococcus cremoris SMQ-461, generating non-EPS-producing reversible mutants. This is the first report of a chromosomal location for EPS genetic elements in Lactococcus cremoris, with novel glycosyltransferases not encountered before in lactic acid bacteria.

scholarly journals Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone

1999 ◽  
Vol 181 (1) ◽  
pp. 338-340 ◽  
Author(s):  
Richard van Kranenburg ◽  
Iris I. van Swam ◽  
Joey D. Marugg ◽  
Michiel Kleerebezem ◽  
Willem M. de Vos
Keyword(s):  
Functional Analysis ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Lactococcus Lactis ◽  
Substrate Specificities ◽  
Exopolysaccharide Biosynthesis

ABSTRACT We used homologous and heterologous expression of the glycosyltransferase genes of the Lactococcus lactis NIZO B40 eps gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.

scholarly journals Engineering of Carbon Distribution between Glycolysis and Sugar Nucleotide Biosynthesis in Lactococcus lactis

2003 ◽  
Vol 69 (2) ◽  
pp. 1129-1135 ◽  
Author(s):  
Ingeborg C. Boels ◽  
Michiel Kleerebezem ◽  
Willem M. de Vos
Keyword(s):  
Lactococcus Lactis ◽  
Metabolic Flux ◽  
Nucleotide Biosynthesis ◽  
Sugar Nucleotides ◽  
Concomitant Reduction ◽  
Sugar Degradation ◽  
Exopolysaccharide Biosynthesis ◽  
Branching Point ◽  
Sugar Phosphates

ABSTRACT We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis. This was realized by using a described isogenic L. lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively. The role of decreased metabolic flux was studied in L. lactis strains with decreased phosphofructokinase activities. The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates. In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels. However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides. Finally, an ∼100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDP-galactose. While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L. lactis.

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

2007 ◽  
Vol 74 (2) ◽  
pp. 424-436 ◽  
Author(s):  
Roland J. Siezen ◽  
Marjo J. C. Starrenburg ◽  
Jos Boekhorst ◽  
Bernadet Renckens ◽  
Douwe Molenaar ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Degradation Products ◽  
Phenotypic Diversity ◽  
Starter Cultures ◽  
Genome Sequences ◽  
Good Correspondence ◽  
Sequence Elements ◽  
Exopolysaccharide Biosynthesis

ABSTRACT 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 α-galactosides, β-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.

Evidence for a shear mechanism for the precipitation of γ-zirconium hydride in zirconium

1978 ◽  
Vol 36 (1) ◽  
pp. 658-659
Author(s):  
G.J.C. Carpenter
Keyword(s):  
Elevated Temperature ◽  
Strain Field ◽  
Zirconium Hydride ◽  
Zirconium Atom ◽  
Atom Diffusion ◽  
Solvus Temperature ◽  
Hexagonal Close Packed ◽  
Partial Dislocations ◽  
The Face

In zirconium-hydrogen alloys, rapid cooling from an elevated temperature causes precipitation of the face-centred tetragonal (fct) phase, γZrH, in the form of needles, parallel to the close-packed <1120>zr directions (1). With low hydrogen concentrations, the hydride solvus is sufficiently low that zirconium atom diffusion cannot occur. For example, with 6 μg/g hydrogen, the solvus temperature is approximately 370 K (2), at which only the hydrogen diffuses readily. Shears are therefore necessary to produce the crystallographic transformation from hexagonal close-packed (hep) zirconium to fct hydride.The simplest mechanism for the transformation is the passage of Shockley partial dislocations having Burgers vectors (b) of the type 1/3<0110> on every second (0001)Zr plane. If the partial dislocations are in the form of loops with the same b, the crosssection of a hydride precipitate will be as shown in fig.1. A consequence of this type of transformation is that a cumulative shear, S, is produced that leads to a strain field in the surrounding zirconium matrix, as illustrated in fig.2a.

Microstructural characterization of a rapidly solidified Al-Fe-V-Si alloy for elevated temperature applications

1988 ◽  
Vol 46 ◽  
pp. 550-551
Author(s):  
R. E. Franck ◽  
J. A. Hawk ◽  
G. J. Shiflet
Keyword(s):  
High Temperature ◽  
Elevated Temperature ◽  
Grain Growth ◽  
Volume Fraction ◽  
Microstructural Characterization ◽  
Second Phase ◽  
Microstructural Stability ◽  
Elevated Temperature Strength ◽  
Temperature Applications

Rapid solidification processing (RSP) is one method of producing high strength aluminum alloys for elevated temperature applications. Allied-Signal, Inc. has produced an Al-12.4 Fe-1.2 V-2.3 Si (composition in wt pct) alloy which possesses good microstructural stability up to 425°C. This alloy contains a high volume fraction (37 v/o) of fine nearly spherical, α-Al12(Fe, V)3Si dispersoids. The improved elevated temperature strength and stability of this alloy is due to the slower dispersoid coarsening rate of the silicide particles. Additionally, the high v/o of second phase particles should inhibit recrystallization and grain growth, and thus reduce any loss in strength due to long term, high temperature annealing.The focus of this research is to investigate microstructural changes induced by long term, high temperature static annealing heat-treatments. Annealing treatments for up to 1000 hours were carried out on this alloy at 500°C, 550°C and 600°C. Particle coarsening and/or recrystallization and grain growth would be accelerated in these temperature regimes.

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