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 The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA2B2 efflux pump-mediated macrolide resistance

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Yuhao Dong ◽  
Qing Li ◽  
Jinzhu Geng ◽  
Qing Cao ◽  
Dan Zhao ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Efflux Pump ◽  
Fold Increase ◽  
Activity Level ◽  
Macrolide Resistance ◽  
Wild Type ◽  
Iron Deprivation ◽  
Pump Activity ◽  
Tonb System ◽  
Increased Sensitivity

AbstractThe TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux.

scholarly journals Characterization, Expression, and Mutation of the Lactococcus lactis galPMKTE Genes, Involved in Galactose Utilization via the Leloir Pathway

2003 ◽  
Vol 185 (3) ◽  
pp. 870-878 ◽  
Author(s):  
Benoît P. Grossiord ◽  
Evert J. Luesink ◽  
Elaine E. Vaughan ◽  
Alain Arnaud ◽  
Willem M. de Vos
Keyword(s):  
Escherichia Coli ◽  
Lactococcus Lactis ◽  
Cell Separation ◽  
Sole Carbon Source ◽  
Wild Type ◽  
Leloir Pathway ◽  
Wild Type Phenotype ◽  
Mutant Strains ◽  
Galactose Utilization ◽  
Long Chain

ABSTRACT 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 galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GalE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the galP, galK, galT, and galE genes was shown by complementation studies performed with both Escherichia coli and L. lactis mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using galP disruption mutant strains of L. lactis MG1363. A galK deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the galE gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the galE mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.

scholarly journals Expression of Six Peptidases fromLactobacillus helveticus in Lactococcus lactis

2001 ◽  
Vol 67 (3) ◽  
pp. 1232-1238 ◽  
Author(s):  
Susanna Luoma ◽  
Kirsi Peltoniemi ◽  
Vesa Joutsjoki ◽  
Terhi Rantanen ◽  
Marja Tamminen ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Fold Increase ◽  
Lactobacillus Helveticus ◽  
Expression Level ◽  
Wild Type ◽  
Activity Measurements ◽  
Low Copy Number ◽  
Genes Encoding ◽  
Dipeptidyl Aminopeptidase ◽  
Proline Iminopeptidase

ABSTRACT For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produceLactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes intoL. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, andpepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepDand pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.

scholarly journals Intestinal mucins from cystic fibrosis mice show increased fucosylation due to an induced Fucα1-2 glycosyltransferase

2002 ◽  
Vol 367 (3) ◽  
pp. 609-616 ◽  
Author(s):  
Kristina A. THOMSSON ◽  
Marina HINOJOSA-KURTZBERG ◽  
Karin A. AXELSSON ◽  
Steven E. DOMINO ◽  
John B. LOWE ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Small Intestine ◽  
Blood Group ◽  
Northern Blot Analysis ◽  
Fold Increase ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Cf Transmembrane Conductance Regulator ◽  
Blood Group H

In gene-targeted mouse models for cystic fibrosis (CF), the disease is mainly manifested by mucus obstruction in the intestine. To explore the mucus composition, mucins insoluble and soluble in 6M guanidinium chloride were purified by three rounds of isopycnic ultracentrifugation from the small and large intestines of CF mice (Cftrm1UNC/Cftrm1UNC) and compared with wild-type mice. The amino acid composition was typical of that for mucins and showed increased amounts of the insoluble (2.5-fold increase) and soluble (7-fold increase) mucins in the small intestine of the CF mice compared with wild-type mice. Mucins from the large intestine of both wild-type and CF mice showed a high but constant level of fucosylation. In contrast, the insoluble and soluble mucins of the small intestine in CF mice revealed a large increase in fucose, whereas those of wild-type mice contained only small amounts of fucose. This increased fucosylation was analysed by releasing the O-linked oligosaccharides followed by GC-MS. NMR spectroscopy revealed that the increased fucosylation was due to an increased expression of blood group H epitopes (Fucα1-2Gal-). Northern-blot analysis, using a probe for the murine Fucα1-2 fucosyltransferase (Fut2), showed an up-regulation of this mRNA in the small intestine of the CF mice, suggesting that this enzyme is responsible for the observed increase in blood group H-type glycosylation. The reason for this up-regulation could be a direct or indirect effect of a non-functional CF transmembrane conductance regulator (CFTR) caused by the absence of CFTR channel.

scholarly journals Specificity Mutants of the Binding Protein of the Oligopeptide Transport System of Lactococcus lactis

2000 ◽  
Vol 182 (6) ◽  
pp. 1600-1608 ◽  
Author(s):  
Antonia Picon ◽  
Edmund R. S. Kunji ◽  
Frank C. Lanfermeijer ◽  
Wil N. Konings ◽  
Bert Poolman
Keyword(s):  
Lactococcus Lactis ◽  
Fold Increase ◽  
Binding Activity ◽  
Kinetic Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Serovar Typhimurium ◽  
Peptide Binding Site ◽  
Oligopeptide Transport

ABSTRACT The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined. To observe the properties of the mutant proteins in vivo, the oppAgene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the oppA gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA fromSalmonella enterica serovar Typhimurium and L. lactis, taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) had a similarKm for transport, whereas theV max was increased more than twofold as compared to the wild-type protein. The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.

Increased Hepatic Fibrinogen Bβ-gene Transcription Is not enough to Increase Plasma Fibrinogen Levels

2001 ◽  
Vol 85 (06) ◽  
pp. 1025-1030 ◽  
Author(s):  
F. Rezaee ◽  
A. Maas ◽  
J. Koopman ◽  
J. H. Verheijen
Keyword(s):  
Transgenic Mice ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Fibrinogen Level ◽  
Fold Increase ◽  
Plasma Fibrinogen ◽  
Cosmid Library ◽  
Chain Gene ◽  
Wild Type

SummaryThe fibrinogen Aα, Bβ, and γ polypeptides are encoded by three separate genes, which are arranged in the order γ-α-β. In order to study the biosynthesis of fibrinogen in vivo we generated a line of transgenic mice carrying extra copies of the fibrinogen β-gene. To clone the mouse fibrinogen Bβ-chain gene, a mouse 129 Sv/Ev genomic cosmid library was screened, using the mouse fibrinogen Aα-, Bγ-chain cDNA. A clone containing the complete fibrinogen Bβ-chain gene including approximately 11-kb of the natural promoter region was identified and subsequently microinjected into mice. Southern blot analysis identified a founder that carried additional copies of the fibrinogen Bβ-chain gene. Transgenic offspring of this founder were interbred and heterozygous and homozygous transgenic mice were obtained. Northern blot analysis demonstrated approximately a 3-fold increase in fibrinogen Bβ mRNA in heterozygous mice as compared to wild-type, whereas homozygous transgenic mice showed approximately a 9-fold increase. The levels of the Aα and γ mRNAs in transgenic homozygous mice were not changed as compared to those in wild-type mice. Fibrinogen levels in plasma were not significantly increased in transgenic mice as compared to wild-type mice.These results indicate that: additional copies of the fibrinogen Bβ-chain gene lead to increased levels of the Bβ-chain mRNA in the liver; the increased levels of Bβ-chain mRNA in homozygous overexpression mice do not change the transcription levels of the two other fibrinogen mRNAs in vivo; the absence of an increased plasma fibrinogen level in the transgenic mice indicates that this level is not regulated solely by transcription of the Bβ-chain gene.

scholarly journals Glyceraldehyde-3-Phosphate Dehydrogenase Has No Control over Glycolytic Flux in Lactococcus lactis MG1363

2003 ◽  
Vol 185 (5) ◽  
pp. 1564-1571 ◽  
Author(s):  
Christian Solem ◽  
Brian J. Koebmann ◽  
Peter R. Jensen
Keyword(s):  
Lactococcus Lactis ◽  
Reference Strain ◽  
Enzyme Level ◽  
Activity Level ◽  
Glycolytic Flux ◽  
Resting Cells ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gapdh Activity ◽  
Absolute Control

ABSTRACT Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has previously been suggested to have almost absolute control over the glycolytic flux in Lactococcus lactis (B. Poolman, B. Bosman, J. Kiers, and W. N. Konings, J. Bacteriol. 169:5887-5890, 1987). Those studies were based on inhibitor titrations with iodoacetate, which specifically inhibits GAPDH, and the data suggested that it should be possible to increase the glycolytic flux by overproducing GAPDH activity. To test this hypothesis, we constructed a series of mutants with GAPDH activities from 14 to 210% of that of the reference strain MG1363. We found that the glycolytic flux was unchanged in the mutants overproducing GAPDH. Also, a decrease in the GAPDH activity had very little effect on the growth rate and the glycolytic flux until 25% activity was reached. Below this activity level, the glycolytic flux decreased proportionally with decreasing GAPDH activity. These data show that GAPDH activity has no control over the glycolytic flux (flux control coefficient = 0.0) at the wild-type enzyme level and that the enzyme is present in excess capacity by a factor of 3 to 4. The early experiments by Poolman and coworkers were performed with cells resuspended in buffer, i.e., nongrowing cells, and we therefore analyzed the control by GAPDH under similar conditions. We found that the glycolytic flux in resting cells was even more insensitive to changes in the GAPDH activity; in this case GAPDH was also present in a large excess and had no control over the glycolytic flux.

scholarly journals Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

1989 ◽  
Vol 9 (4) ◽  
pp. 1507-1512 ◽  
Author(s):  
H Zhu ◽  
H Conrad-Webb ◽  
X S Liao ◽  
P S Perlman ◽  
R A Butow
Keyword(s):  
Genetic Analysis ◽  
Rna Processing ◽  
Fold Increase ◽  
Functional Expression ◽  
Rrna Gene ◽  
Yeast Mitochondria ◽  
Wild Type ◽  
Site Specific ◽  
Var1 Gene ◽  
A Site

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

scholarly journals Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 81-95 ◽  
Author(s):  
E J Louis ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Stop Codon ◽  
Fold Increase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nonsense Mutations ◽  
Chromosome Iii ◽  
Meiosis I ◽  
Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

Growth, nisA Gene Expression, and In Situ Activity of Novel Lactococcus lactis subsp. cremoris Costarter Culture in Commercial Hard Cheese Production

2017 ◽  
Vol 80 (12) ◽  
pp. 2137-2146 ◽  
Author(s):  
Dimitrios Noutsopoulos ◽  
Athanasia Kakouri ◽  
Eleftheria Kartezini ◽  
Dimitrios Pappas ◽  
Efstathios Hatziloukas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lactococcus Lactis ◽  
Starter Culture ◽  
Fold Increase ◽  
Raw Milk ◽  
Good Growth ◽  
Lactococcus Lactis Subsp ◽  
Quantitative Expression ◽  
Hard Cheese

ABSTRACT This study evaluated in situ expression of the nisA gene by an indigenous, nisin A–producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A–mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

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