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M Kleerebezem

Publications and source records attributed to M Kleerebezem.

At least 19 recordsLinked to original sources
PubMed2006

DNA micro-array-based identification of bile-responsive genes in Lactobacillus plantarum.

AIMS: The purpose of this study was to determine the global transcriptional response in a food-associated lactic acid bacterium during bile stress. METHODS AND RESULTS: Clone-based DNA micro-arrays were employed to describe the global transcriptional response of Lactobacillus plantarum WCFS1 towards 0.1% porcine bile. Comparison of differential transcript profiles obtained during growth of Lact. plantarum on plates with and without bile revealed 28 and 62 putative genes, of which the expression was at least 2.5-fold up- or down-regulated by bile, respectively. Approximately, 50% of these genes appeared genetically linked, and 12 bile-responsive gene clusters were identified. Seven of the identified bile-responsive genes and gene clusters encode typical stress-related functions, including glutathione reductase and glutamate decarboxylase, involved in oxidative and acid stress, respectively. Moreover, 14 bile-responsive genes and gene clusters were identified that encode proteins that are located in the cell envelope, including the dlt operon and the F1F0 ATPase. CONCLUSIONS: The identification of a relatively high number of genes encoding cell envelope functions indicates a major impact of bile acids on the integrity and/or functionality of the cytoplasmic membrane and cell wall. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here provide valuable clues towards the defence mechanisms that play a role during bile stress in Lact. plantarum.

Animals↗
PubMed2002

Metabolic engineering of acetaldehyde production by Streptococcus thermophilus.

The process of acetaldehyde formation by the yogurt bacterium Streptococcus thermophilus is described in this paper. Attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the glyA gene. In S. thermophilus, SHMT also possesses threonine aldolase (TA) activity, the interconversion of threonine into glycine and acetaldehyde. In this work, several wild-type S. thermophilus strains were screened for acetaldehyde production in the presence and absence of L-threonine. Supplementation of the growth medium with L-threonine led to an increase in acetaldehyde production. Furthermore, acetaldehyde formation during fermentation could be correlated to the TA activity of SHMT. To study the physiological role of SHMT, a glyA mutant was constructed by gene disruption. Inactivation of glyA resulted in a severe reduction in TA activity and complete loss of acetaldehyde formation during fermentation. Subsequently, an S. thermophilus strain was constructed in which the glyA gene was cloned under the control of a strong promoter (P(LacA)). When this strain was used for fermentation, an increase in TA activity and in acetaldehyde and folic acid production was observed. These results show that, in S. thermophilus, SHMT, displaying TA activity, constitutes the main pathway for acetaldehyde formation under our experimental conditions. These findings can be used to control and improve acetaldehyde production in fermented (dairy) products with S. thermophilus as starter culture.

Acetaldehyde↗
PubMed2001

Peptide pheromone-dependent regulation of antimicrobial peptide production in Gram-positive bacteria: a case of multicellular behavior.

Quorum sensing enables unicellular organisms to behave in a multicellular way by allowing population-wide synchronized adaptive responses that involve modulation of a wide range of physiological responses in a cell density-, cell proximity- or growth phase-dependent manner. Examples of processes modulated by quorum sensing are the development of genetic competence, conjugative plasmid transfer, sporulation and cell differentiation, biofilm formation, virulence response, production of antibiotics, antimicrobial peptides and toxins, and bioluminescence (for reviews see [38]). The cell-to-cell communication strategies involved in these processes are based on the utilization of small signal molecules produced and released into the environment by the microorganisms. These communication molecules are referred to as pheromones and act as chemical messengers that transmit information across space. The extracellular pheromones accumulate in the environment and trigger a response in the target cells when its concentration reaches a certain threshold value. Elucidation of the chemical nature of the pheromones modulating the processes mentioned above reveals that most of them are unmodified peptides, post-translationally modified peptides, N-acyl homoserine lactones, or butyrolactones. Lactone-based pheromones are the preferred communication signals in Gram-negative bacteria (for review see [47,48]), whereas peptide-based pheromones are the predominant extracellular signals among Gram-positive bacteria (for review see [37,61]). However, lactone-based pheromones are utilized as signals that modulate differentiation and secondary metabolism production in Streptomyces (for review see [20]). This review focuses on the major advances and current views of the peptide-pheromone dependent regulatory circuits involved in production of antimicrobial peptides in Gram-positive bacteria.

Anti-Bacterial Agents↗
PubMed2001

A two-component signal-transduction cascade in Carnobacterium piscicola LV17B: two signaling peptides and one sensor-transmitter.

In the lactic acid bacterium Carnobacterium piscicola LV17B a peptide-pheromone dependent quorum-sensing mode is involved in the regulation of bacteriocin production. Bacteriocin CB2 was identified as an environmental signal that induces bacteriocin production. Here, we demonstrate that a second 24 amino acid peptide (CS) also induces bacteriocin production. Transcription activation of several carnobacteriocin operons is triggered by CB2 or CS via a two-component signal transduction system composed of CbnK and CbnR.

Bacterial Proteins↗
PubMed2001

Functional analysis of the Lactococcus lactis galU and galE genes and their impact on sugar nucleotide and exopolysaccharide biosynthesis.

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.

Cloning, Molecular↗
PubMed2001

Hierarchical control versus autoregulation of carbohydrate utilization in bacteria.

The involvement of phosphoeno/pyruvate:sugar phosphotransferase (PTS) proteins, like HPr and IIA(Glc), in the regulation of carbohydrate utilization has been well established in Gram-negative and Gram-positive bacteria. The majority of the studies of PTS-mediated regulation have been concerned with the hierarchical control of carbohydrate utilization, which results in the preferential utilization of a particular carbohydrate from a mixture of substrates. The underlying mechanisms of PTS-mediated hierarchical control involve the inhibition of expression of other catabolic enzymes and transporters and/or the allosteric regulation of their activity, which prevents the transcriptional inducer to be formed or taken up into the cell. More recently, it has become clear that PTS components allow also the cell to tune the uptake rate(s) to the carbohydrate availability in the medium and the metabolic capacity of the cell. The different phosphorylated species of HPr play a central role in this autoregulatory control circuit, both at the gene and at the protein level. Our knowledge of hierarchical control and autoregulation of carbohydrate utilization in bacteria is discussed.

Bacteria↗
PubMed2000

Nucleotide sequence analysis of the lactococcal EPS plasmid pNZ4000.

The complete 42180-bp nucleotide sequence of the mobilization plasmid pNZ4000, coding for exopolysaccharide (EPS) production in Lactococcus lactis, was determined. This plasmid contains a region involved in EPS biosynthesis, four functional replicons, a region containing mobilization genes, and three origin of transfer (oriT) sequences. Sequences identical to these oriT sequences were also found on two other lactococcal plasmids and a plasmid from Lactobacillus helveticus. Several complete and partial IS elements were identified on pNZ4000, including iso-ISS1, iso-IS946, and iso-IS982 sequences. Furthermore, pNZ4000 contains a gene cluster that may encode a cobalt transport system and a gene that encodes a CorA homologue which may function as a magnesium transporter.

Amino Acid Sequence↗
PubMed2000

Improved vectors for nisin-controlled expression in gram-positive bacteria.

A set of shuttle vectors, able to replicate in Escherichia coli and in gram-positive bacteria, containing a nisin-inducible promoter (PnisA) and genes encoding NisR and NisK, the two-component signaling mechanism for activating transcription from PnisA in the presence of nisin, was constructed. To test these vectors, Enterococcus faecalis pCF10 plasmid genes prgX, prgY, and prgZ, which respectively encode cytosolic, integral membrane, and cell surface proteins, were cloned downstream of PnisA. Increased protein expression, in the presence of nisin, was demonstrated by Western blot analysis.

Bacterial Proteins↗
PubMed2000

Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects.

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (beta-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.

Animals↗
PubMed2000

Lactococcus lactis as a cell factory for high-level diacetyl production.

We report the engineering of Lactococcus lactis for the efficient conversion of sugar into diacetyl by combining NADH-oxidase overproduction and alpha-acetolactate decarboxylase inactivation. Eighty percent of the carbon flux was found to be rerouted via alpha-acetolactate to the production of diacetyl by preloading the cells with NADH-oxidase before their use as a cell factory.

Carboxy-Lyases↗
PubMed2000

Control of lactose transport, beta-galactosidase activity, and glycolysis by CcpA in Streptococcus thermophilus: evidence for carbon catabolite repression by a non-phosphoenolpyruvate-dependent phosphotransferase system sugar.

Streptococcus thermophilus, unlike many other gram-positive bacteria, prefers lactose over glucose as the primary carbon and energy source. Moreover, lactose is not taken up by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) but by the dedicated transporter LacS. In this paper we show that CcpA plays a crucial role in the fine-tuning of lactose transport, beta-galactosidase (LacZ) activity, and glycolysis to yield optimal glycolytic flux and growth rate. A catabolite-responsive element (cre) was identified in the promoter of the lacSZ operon, indicating a possible role for regulation by CcpA. Transcriptional analysis showed a sevenfold relief of repression in the absence of a functional CcpA when cells were grown on lactose. This CcpA-mediated repression of lacSZ transcription did not occur in wild-type cells during growth on galactose, taken up by the same LacS transport system. Lactose transport during fermentation was increased significantly in strains carrying a disrupted ccpA gene. Moreover, a ccpA disruption strain was found to release substantial amounts of glucose into the medium when grown on lactose. Transcriptional analysis of the ldh gene showed that expression was induced twofold during growth on lactose compared to glucose or galactose, in a CcpA-dependent manner. A reduced rate of glycolysis concomitant with an increased lactose transport rate could explain the observed expulsion of glucose in a ccpA disruption mutant. We propose that CcpA in S. thermophilus acts as a catabolic regulator during growth on the preferred non-PTS sugar lactose. In contrast to other bacteria, S. thermophilus possesses an overcapacity for lactose uptake that is repressed by CcpA to match the rate-limiting glycolytic flux.

Bacterial Proteins↗
PubMed1999

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

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.

Glycolysis↗
PubMed1999

Metabolic engineering of lactic acid bacteria: overview of the approaches and results of pathway rerouting involved in food fermentations.

Lactic acid bacteria such as Lactococcus lactis are the microorganisms of choice for performing metabolic engineering in relation to food fermentation. These bacteria are used extensively in food fermentations, they have a simple and therefore controllable metabolism and the molecular genetics of these food bacteria is well-developed. There have been recent successes in metabolic engineering in these lactic acid bacteria, including examples of changes in both primary metabolism (diacetyl and alanine) and secondary metabolism (exopolysaccharides and flavour).

Alanine↗
PubMed1999

Genetics and engineering of microbial exopolysaccharides for food: approaches for the production of existing and novel polysaccharides.

Microbial exopolysaccharides (EPSs) are used in the food industry for their unique properties as viscosifiers, stabilisers, emulsifiers or gelling agents. In recent years, significant progress in the understanding of the genetics and biochemistry of microbial EPS synthesis by both Gram-negative and Gram-positive bacteria has been made. Biosynthesis pathways have been elucidated, and several of the genes involved have been characterised. This knowledge can now be applied to start EPS engineering or to improve EPS production.

Amino Acid Sequence↗
PubMed1999

Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering.

We report the engineering of Lactococcus lactis to produce the amino acid L-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH-->L-lactate + NAD+) is performed by L-lactate dehydrogenase (L-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (L-AlaDH; pyruvate + NADH + NH4+ -->L-alanine + NAD+ + H2O). Expression of L-AlaDH in an L-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.

Alanine↗
PubMed1999

Regulation of exopolysaccharide production by Lactococcus lactis subsp. cremoris By the sugar source.

Lactococcus lactis produced more exopolysaccharide (EPS) on glucose than on fructose as the sugar substrate, although the transcription level of the eps gene cluster was independent of the sugar source. A major difference between cells grown on the two substrates was the capacity to produce sugar nucleotides, the EPS precursors. However, the activities of the enzymes required for the synthesis of nucleotide sugars were not changed upon growth on different sugars. The activity of fructosebisphosphatase (FBPase) was by far the lowest of the enzymes involved in precursor formation under all conditions. FBPase catalyzes the conversion of fructose-1, 6-diphosphate into fructose-6-phosphate, which is an essential step in the biosynthesis of sugar nucleotides from fructose but not from glucose. By overexpression of the fbp gene, which resulted in increased EPS synthesis on fructose, it was proven that the low activity of FBPase is indeed limiting not only for EPS production but also for growth on fructose as a sugar source.

Cell Wall↗
PubMed1999

Exopolysaccharide biosynthesis in Lactococcus lactis NIZO B40: functional analysis of the glycosyltransferase genes involved in synthesis of the polysaccharide backbone.

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.

Base Sequence↗