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Papers by Jan Martinussen

Research paper thumbnail of The growth rate of pyrimidine auxotrophic mutants of Lactococcus lactis MG1363 is reduced in the presence of exogenous aspartate

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Research paper thumbnail of pyrimidine biosynthesis. including a novel gene, pyrK, involved in pyrKDbF operon from Lactococcus lactis Sequence analysis and identification of the

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Research paper thumbnail of Characterization of the molecular and genetic regulation of the nucleotide metabolism in Lactococcus lactis

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Research paper thumbnail of The orotate transporter oroP from Lactococcus lactis can be used both as a very efficient, food-grade selection and counter-selection marker for strain construction in many different organisms

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Research paper thumbnail of Analysis of plasmid pDBORO – A plasmid encoding an orotate transporter from Lactococcus lactis subsp. lactis biovar. diacetylactis strain DB0410

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Research paper thumbnail of {"__content__"=>"Interplay Between Capsule Expression and Uracil Metabolism in D39.", "i"=>{"__content__"=>"Streptococcus pneumoniae"}}

Frontiers in microbiology, 2018

Pyrimidine nucleotides play an important role in the biosynthesis of activated nucleotide sugars ... more Pyrimidine nucleotides play an important role in the biosynthesis of activated nucleotide sugars (NDP-sugars). NDP-sugars are the precursors of structural polysaccharides in bacteria, including capsule, which is a major virulence factor of the human pathogen . In this work, we identified a spontaneous non-reversible mutant of strain D39 that displayed a non-producing capsule phenotype. Whole-genome sequencing analysis of this mutant revealed several non-synonymous single base modifications, including in genes of the synthesis of pyrimidines and in the -10 box of capsule operon promoter (P). By directed mutagenesis we showed that the point mutation in P was solely responsible for the drastic decrease in capsule expression. We also demonstrated that D39 subjected to uracil deprivation shows increased biomass and decreased P activity and capsule amounts. Importantly, P expression is further decreased by mutating the first gene of the synthesis of pyrimidines, . In contrast, the absence...

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Research paper thumbnail of Metabolic characterization and transformation of the non-dairy Lactococcus lactis strain KF147, for production of ethanol from xylose

Biotechnology journal, Jan 18, 2017

The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energ... more The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, we characterized growth and product formation for KF147 when grown on xylose. In a defined medium KF147 was found to co-metabolize xylose and arginine, resulting in bi-phasic growth. Especially at low xylose concentrations, arginine significantly improved growth rate. To facilitate further studies of the xylose metabolism, we eliminated arginine catabolism by deleting the arcA gene encoding the arginine deiminase. The fermentation product profile suggested two routes for xylose degradation, the phosphoketolase pathway and the pentose phosphate pathway. Inactivation of the phosphoketolase pathway redirected the entire flux through the pentose phosphate pathway whereas over-expression of phosphoketolase increased the flux through the phosphoketolase pathway. I...

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Research paper thumbnail of Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

Microbiology and molecular biology reviews : MMBR, Mar 1, 2017

Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is sy... more Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of ...

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Research paper thumbnail of The ribosome dimerization factor YfiA is the major protein synthesized after abrupt glucose depletion in <i>Lactococcus lactis</i>

Microbiology, 2016

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Research paper thumbnail of Orotate transporter encoding maker genes

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Research paper thumbnail of Addition of ?-ketoglutarate enhances formation of volatiles by Staphylococcus carnosus during sausage fermentation

Meat Sci, 2004

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Research paper thumbnail of Two different nucleoside transporters in Lactococcus lactis with different substrate specificities

Microbiology Sgm, 2010

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Research paper thumbnail of Orotate Transporter Encoding Marker Genes

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Research paper thumbnail of The CytR repressor antagonized cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

Journal of Bacteriology

We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor... more We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) CytR selectively regulated cAMP-CRP-dependent initiations, although transcription started from the same site in deoCp2 in the absence or presence of cAMP-CRP; (ii) deletion of the uppermost cAMP-CRP target (CRP-2) resulted in loss of CytR regulation, but had only a minor effect on positive control by the cAMP-CRP complex; (iii) introduction of point mutations in either CRP target resulted in loss of CytR regulation; and (iv) regulation by CytR of deletion mutants lacking CRP-2 could be specifically reestablished by increasing the intracellular concentration of CytR. These findings indicate that both CRP targets are required for efficient CytR repression of deoCp2. Models for the action of Cyt...

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Research paper thumbnail of Sequence Analysis and Identification of thepyrKDbFOperon from Lactococcus lactisIncluding a Novel Gene,pyrK, Involved in Pyrimidine Biosynthesis

Three genes encoding enzymes involved in the biosynthesis of pyrimidines have been found to const... more Three genes encoding enzymes involved in the biosynthesis of pyrimidines have been found to constitute an operon inLactococcus lactis. Two of the genes are the well-knownpyrgenespyrDbandpyrF, encoding dihydro- orotate dehydrogenase and orotidine monophosphate decarboxylase, respectively. The third gene encodes a protein which was shown to be necessary for the activity of thepyrDb-encoded dihydroorotate dehydrogenase; we propose to name the genepyrK.

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Research paper thumbnail of Bistability in a Metabolic Network Underpins the De Novo Evolution of Colony Switching in Pseudomonas fluorescens

PLoS biology, 2015

Phenotype switching is commonly observed in nature. This prevalence has allowed the elucidation o... more Phenotype switching is commonly observed in nature. This prevalence has allowed the elucidation of a number of underlying molecular mechanisms. However, little is known about how phenotypic switches arise and function in their early evolutionary stages. The first opportunity to provide empirical insight was delivered by an experiment in which populations of the bacterium Pseudomonas fluorescens SBW25 evolved, de novo, the ability to switch between two colony phenotypes. Here we unravel the molecular mechanism behind colony switching, revealing how a single nucleotide change in a gene enmeshed in central metabolism (carB) generates such a striking phenotype. We show that colony switching is underpinned by ON/OFF expression of capsules consisting of a colanic acid-like polymer. We use molecular genetics, biochemical analyses, and experimental evolution to establish that capsule switching results from perturbation of the pyrimidine biosynthetic pathway. Of central importance is a bifur...

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Research paper thumbnail of The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

Journal of bacteriology, 1990

We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor... more We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) CytR selectively regulated cAMP-CRP-dependent initiations, although transcription started from the same site in deoCp2 in the absence or presence of cAMP-CRP; (ii) deletion of the uppermost cAMP-CRP target (CRP-2) resulted in loss of CytR regulation, but had only a minor effect on positive control by the cAMP-CRP complex; (iii) introduction of point mutations in either CRP target resulted in loss of CytR regulation; and (iv) regulation by CytR of deletion mutants lacking CRP-2 could be specifically reestablished by increasing the intracellular concentration of CytR. These findings indicate that both CRP targets are required for efficient CytR repression of deoCp2. Models for the action of Cyt...

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Research paper thumbnail of The hok killer gene family in gram-negative bacteria

The New biologist, 1990

The seven members of the hok killer gene family in Gram-negative bacteria are described here. The... more The seven members of the hok killer gene family in Gram-negative bacteria are described here. The members of this gene family have been sequenced and include hok/sok from plasmid R1, flm and srnB from plasmid F, pnd from plasmids R483 and R16, and gef and relF, which are located on the Escherichia coli chromosome. The killer proteins encoded by these loci are highly toxic polypeptides of 50 to 52 amino acids. The proteins kill the cells from the inside by interfering with a vital function in the cell membrane. On the basis of their relatedness, the killer proteins and their corresponding loci are divided into four subfamilies. The members of one subfamily, hok/sok and flm, mediate plasmid maintenance by killing plasmid-free cells. The pnd and srnB subfamilies were discovered through their abilities to cause membrane damage and degradation of stable RNA. gef and relF, which constitute the chromosomal subfamily, were found because of their sequence similarity at the DNA and protein le...

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Research paper thumbnail of Multi-stress resistance in Lactococcus lactis is actually escape from purine-induced stress sensitivity

Microbiology (Reading, England), 2014

Multi-stress resistance is a widely documented and fascinating phenotype of lactococci where sing... more 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 show...

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Research paper thumbnail of Cloning and verification of the Lactococcus lactis pyrG gene and characterization of the gene product, CTP synthase

The Journal of biological chemistry, Jan 12, 2001

The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and s... more The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactis strains harboring disrupted pyrG alleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrG product is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrG mutant could be constructed in the presence of a functional cdd gene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mm for dUTP makes it unlikely that this reaction plays a significant physiological role. As for...

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Research paper thumbnail of The growth rate of pyrimidine auxotrophic mutants of Lactococcus lactis MG1363 is reduced in the presence of exogenous aspartate

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Research paper thumbnail of pyrimidine biosynthesis. including a novel gene, pyrK, involved in pyrKDbF operon from Lactococcus lactis Sequence analysis and identification of the

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Characterization of the molecular and genetic regulation of the nucleotide metabolism in Lactococcus lactis

Bookmarks Related papers MentionsView impact

Research paper thumbnail of The orotate transporter oroP from Lactococcus lactis can be used both as a very efficient, food-grade selection and counter-selection marker for strain construction in many different organisms

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Analysis of plasmid pDBORO – A plasmid encoding an orotate transporter from Lactococcus lactis subsp. lactis biovar. diacetylactis strain DB0410

Bookmarks Related papers MentionsView impact

Research paper thumbnail of {"__content__"=>"Interplay Between Capsule Expression and Uracil Metabolism in D39.", "i"=>{"__content__"=>"Streptococcus pneumoniae"}}

Frontiers in microbiology, 2018

Pyrimidine nucleotides play an important role in the biosynthesis of activated nucleotide sugars ... more Pyrimidine nucleotides play an important role in the biosynthesis of activated nucleotide sugars (NDP-sugars). NDP-sugars are the precursors of structural polysaccharides in bacteria, including capsule, which is a major virulence factor of the human pathogen . In this work, we identified a spontaneous non-reversible mutant of strain D39 that displayed a non-producing capsule phenotype. Whole-genome sequencing analysis of this mutant revealed several non-synonymous single base modifications, including in genes of the synthesis of pyrimidines and in the -10 box of capsule operon promoter (P). By directed mutagenesis we showed that the point mutation in P was solely responsible for the drastic decrease in capsule expression. We also demonstrated that D39 subjected to uracil deprivation shows increased biomass and decreased P activity and capsule amounts. Importantly, P expression is further decreased by mutating the first gene of the synthesis of pyrimidines, . In contrast, the absence...

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Research paper thumbnail of Metabolic characterization and transformation of the non-dairy Lactococcus lactis strain KF147, for production of ethanol from xylose

Biotechnology journal, Jan 18, 2017

The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energ... more The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, we characterized growth and product formation for KF147 when grown on xylose. In a defined medium KF147 was found to co-metabolize xylose and arginine, resulting in bi-phasic growth. Especially at low xylose concentrations, arginine significantly improved growth rate. To facilitate further studies of the xylose metabolism, we eliminated arginine catabolism by deleting the arcA gene encoding the arginine deiminase. The fermentation product profile suggested two routes for xylose degradation, the phosphoketolase pathway and the pentose phosphate pathway. Inactivation of the phosphoketolase pathway redirected the entire flux through the pentose phosphate pathway whereas over-expression of phosphoketolase increased the flux through the phosphoketolase pathway. I...

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Research paper thumbnail of Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

Microbiology and molecular biology reviews : MMBR, Mar 1, 2017

Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is sy... more Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of ...

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Research paper thumbnail of The ribosome dimerization factor YfiA is the major protein synthesized after abrupt glucose depletion in <i>Lactococcus lactis</i>

Microbiology, 2016

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Research paper thumbnail of Orotate transporter encoding maker genes

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Addition of ?-ketoglutarate enhances formation of volatiles by Staphylococcus carnosus during sausage fermentation

Meat Sci, 2004

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Two different nucleoside transporters in Lactococcus lactis with different substrate specificities

Microbiology Sgm, 2010

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Orotate Transporter Encoding Marker Genes

Bookmarks Related papers MentionsView impact

Research paper thumbnail of The CytR repressor antagonized cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

Journal of Bacteriology

We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor... more We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) CytR selectively regulated cAMP-CRP-dependent initiations, although transcription started from the same site in deoCp2 in the absence or presence of cAMP-CRP; (ii) deletion of the uppermost cAMP-CRP target (CRP-2) resulted in loss of CytR regulation, but had only a minor effect on positive control by the cAMP-CRP complex; (iii) introduction of point mutations in either CRP target resulted in loss of CytR regulation; and (iv) regulation by CytR of deletion mutants lacking CRP-2 could be specifically reestablished by increasing the intracellular concentration of CytR. These findings indicate that both CRP targets are required for efficient CytR repression of deoCp2. Models for the action of Cyt...

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Research paper thumbnail of Sequence Analysis and Identification of thepyrKDbFOperon from Lactococcus lactisIncluding a Novel Gene,pyrK, Involved in Pyrimidine Biosynthesis

Three genes encoding enzymes involved in the biosynthesis of pyrimidines have been found to const... more Three genes encoding enzymes involved in the biosynthesis of pyrimidines have been found to constitute an operon inLactococcus lactis. Two of the genes are the well-knownpyrgenespyrDbandpyrF, encoding dihydro- orotate dehydrogenase and orotidine monophosphate decarboxylase, respectively. The third gene encodes a protein which was shown to be necessary for the activity of thepyrDb-encoded dihydroorotate dehydrogenase; we propose to name the genepyrK.

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Research paper thumbnail of Bistability in a Metabolic Network Underpins the De Novo Evolution of Colony Switching in Pseudomonas fluorescens

PLoS biology, 2015

Phenotype switching is commonly observed in nature. This prevalence has allowed the elucidation o... more Phenotype switching is commonly observed in nature. This prevalence has allowed the elucidation of a number of underlying molecular mechanisms. However, little is known about how phenotypic switches arise and function in their early evolutionary stages. The first opportunity to provide empirical insight was delivered by an experiment in which populations of the bacterium Pseudomonas fluorescens SBW25 evolved, de novo, the ability to switch between two colony phenotypes. Here we unravel the molecular mechanism behind colony switching, revealing how a single nucleotide change in a gene enmeshed in central metabolism (carB) generates such a striking phenotype. We show that colony switching is underpinned by ON/OFF expression of capsules consisting of a colanic acid-like polymer. We use molecular genetics, biochemical analyses, and experimental evolution to establish that capsule switching results from perturbation of the pyrimidine biosynthetic pathway. Of central importance is a bifur...

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Research paper thumbnail of The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

Journal of bacteriology, 1990

We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor... more We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) CytR selectively regulated cAMP-CRP-dependent initiations, although transcription started from the same site in deoCp2 in the absence or presence of cAMP-CRP; (ii) deletion of the uppermost cAMP-CRP target (CRP-2) resulted in loss of CytR regulation, but had only a minor effect on positive control by the cAMP-CRP complex; (iii) introduction of point mutations in either CRP target resulted in loss of CytR regulation; and (iv) regulation by CytR of deletion mutants lacking CRP-2 could be specifically reestablished by increasing the intracellular concentration of CytR. These findings indicate that both CRP targets are required for efficient CytR repression of deoCp2. Models for the action of Cyt...

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Research paper thumbnail of The hok killer gene family in gram-negative bacteria

The New biologist, 1990

The seven members of the hok killer gene family in Gram-negative bacteria are described here. The... more The seven members of the hok killer gene family in Gram-negative bacteria are described here. The members of this gene family have been sequenced and include hok/sok from plasmid R1, flm and srnB from plasmid F, pnd from plasmids R483 and R16, and gef and relF, which are located on the Escherichia coli chromosome. The killer proteins encoded by these loci are highly toxic polypeptides of 50 to 52 amino acids. The proteins kill the cells from the inside by interfering with a vital function in the cell membrane. On the basis of their relatedness, the killer proteins and their corresponding loci are divided into four subfamilies. The members of one subfamily, hok/sok and flm, mediate plasmid maintenance by killing plasmid-free cells. The pnd and srnB subfamilies were discovered through their abilities to cause membrane damage and degradation of stable RNA. gef and relF, which constitute the chromosomal subfamily, were found because of their sequence similarity at the DNA and protein le...

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Research paper thumbnail of Multi-stress resistance in Lactococcus lactis is actually escape from purine-induced stress sensitivity

Microbiology (Reading, England), 2014

Multi-stress resistance is a widely documented and fascinating phenotype of lactococci where sing... more 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 show...

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Research paper thumbnail of Cloning and verification of the Lactococcus lactis pyrG gene and characterization of the gene product, CTP synthase

The Journal of biological chemistry, Jan 12, 2001

The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and s... more The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactis strains harboring disrupted pyrG alleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrG product is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrG mutant could be constructed in the presence of a functional cdd gene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mm for dUTP makes it unlikely that this reaction plays a significant physiological role. As for...

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