Biomedical subjects

M Kleerebezem

Publications and source records attributed to M Kleerebezem.

33 records · Page 2Linked to original sources

PubMed1999

Acetate utilization in Lactococcus lactis deficient in lactate dehydrogenase: a rescue pathway for maintaining redox balance.

Acetate was shown to improve glucose fermentation in Lactococcus lactis deficient in lactate dehydrogenase. 13C and 1H nuclear magnetic resonance studies using [2-13C]glucose and [2-(13)C]acetate as substrates demonstrated that acetate was exclusively converted to ethanol. This novel pathway provides an alternative route for NAD+ regeneration in the absence of lactate dehydrogenase.

Acetates↗

PubMed1999

Functional analysis of glycosyltransferase genes from Lactococcus lactis and other gram-positive cocci: complementation, expression, and diversity.

Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the eps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the eps gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene epsD was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This epsD deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from L. lactis or Streptococcus thermophilus, complementation with priming glucosyltransferase genes involved in L. lactis EPS and Streptococcus pneumoniae capsule biosynthesis could completely restore or even increase EPS production in L. lactis.

Amino Acid Sequence↗

PubMed1998

Use of the lactococcal nisA promoter to regulate gene expression in gram-positive bacteria: comparison of induction level and promoter strength.

We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac, xylA, and lacA promoters in B. subtilis and in S. pyogenes. Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.

Bacillus subtilis↗

PubMed1998

Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase.

NADH oxidase-overproducing Lactococcus lactis strains were constructed by cloning the Streptococcus mutans nox-2 gene, which encodes the H2O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the L. lactis nisA promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via alpha-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD+ ratios in the cells.

Acetoin↗

PubMed1997

Role of the carboxy-terminal phenylalanine in the biogenesis of outer membrane protein PhoE of Escherichia coli K-12.

Most bacterial outer membrane proteins contain a phenylalanine at their C terminus. It has been shown that this residue has an important role in the efficient and correct assembly of PhoE protein into the Escherichia coli outer membrane, since its substitution or deletion resulted in the accumulation of trypsin-sensitive monomers of this normally trimeric protein. Here, the role of the C-terminal Phe in the assembly of PhoE was studied in further detail. Immunocytochemical labelling on ultrathin cryosections revealed that a mutant PhoE protein that lacks the C-terminal Phe accumulates in the periplasm. However, when the expression levels of the altered species were reduced, the efficiency of outer membrane incorporation was increased and the lethal effects were alleviated. The role of the C-terminal Phe in protein folding, trimerization and outer membrane incorporation was further studied in vitro. Deletion of this residue interfered with the efficiency of the formation of an assembly-competent folded monomer, and the stability of this PhoE form was affected. The in vitro trimerization and insertion into outer membranes were not affected by the mutation.

Bacterial Proteins↗

PubMed1997

Controlled overproduction of proteins by lactic acid bacteria.

Lactic acid bacteria are widely used in industrial food fermentations, contributing to flavour, texture and preservation of the fermented products. Here we describe recent advances in the development of controlled gene expression systems, which allow the regulated overproduction of any desirable protein by lactic acid bacteria. Some systems benefit from the fact that the expression vectors, marker genes and inducing factors can be used directly in food applications since they are all derived from food-grade lactic acid bacteria. These systems have also been employed for the development of autolytic bacteria, suitable for various industrial applications.

Biotechnology↗

PubMed1997

Expression systems for industrial Gram-positive bacteria with low guanine and cytosine content.

Recent years have seen an increase in the development of gene expression systems for industrial Gram-positive bacteria with low guanine and cytosine content that belong to the genera Bacillus, Clostridium, Lactococcus, Lactobacillus, Staphylococcus and Streptococcus. In particular, considerable advances have been made in the construction of inducible gene expression systems based on the capacity of these bacteria to utilize specific sugars or to secrete autoinducing peptides that are involved in quorum sensing. These controlled expression systems allow for present and future exploitation of these bacteria as cell factories in medical, agricultural, and food biotechnology.

Biotechnology↗

PubMed1997

Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria.

Cell-density-dependent gene expression appears to be widely spread in bacteria. This quorum-sensing phenomenon has been well established in Gram-negative bacteria, where N-acyl homoserine lactones are the diffusible communication molecules that modulate cell-density-dependent phenotypes. Similarly, a variety of processes are known to be regulated in a cell-density- or growth-phase-dependent manner in Gram-positive bacteria. Examples of such quorum-sensing modes in Gram-positive bacteria are the development of genetic competence in Bacillus subtilis and Streptococcus pneumoniae, the virulence response in Staphylococcus aureus, and the production of antimicrobial peptides by several species of Gram-positive bacteria including lactic acid bacteria. Cell-density-dependent regulatory modes in these systems appear to follow a common theme, in which the signal molecule is a post-translationally processed peptide that is secreted by a dedicated ATP-binding-cassette exporter. This secreted peptide pheromone functions as the input signal for a specific sensor component of a two-component signal-transduction system. Moreover, genetic linkage of the common elements involved results in autoregulation of peptide-pheromone production.

Amino Acid Sequence↗

PubMed1997

Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lactococcus lactis MG1363, Leuconostoc lactis NZ6091, and Lactobacillus helveticus CNRZ32. Typically, the beta-glucuronidase activity (used as a reporter in this study) remained below the detection limits under noninducing conditions and could be raised to high levels, by addition of subinhibitory amounts of nisin to the growth medium, while exhibiting a linear dose-response relationship. These results demonstrate that the nisin-inducible system can be functionally implemented in lactic acid bacteria other than Lactococcus lactis.

Gene Expression Regulation, Bacterial↗

PubMed1997

Characterization of a locus from Carnobacterium piscicola LV17B involved in bacteriocin production and immunity: evidence for global inducer-mediated transcriptional regulation.

Mutational, nucleotide sequence, and transcriptional analyses of a 10-kb fragment (carnobacteriocin locus) from the 61-kb plasmid of Carnobacterium piscicola LV17B demonstrated the presence of two gene clusters (cbnXY and cbnSKRTD) upstream of the previously sequenced carnobacteriocin B2 structural and immunity genes (cbnB2 and cbiB2). Deduced products of cbnK and cbnR have sequence similarity to proteins of Agr-type two-component signal transduction systems, and those of cbnT and cbnD have sequence similarity to proteins of signal sequence-independent secretion systems. Deduced products of cbnX, cbnY, and cbnS are class II-type bacteriocin precursors with potential leader peptides containing double-glycine cleavage sites. Genetic analysis indicated that the 10-kb locus contains information required for the production of, and immunity to, the plasmid-encoded carnobacteriocin B2 and the chromosomally encoded carnobacteriocin BM1. In addition, this locus is involved in the production of at least one additional antimicrobial compound and an inducer factor that plays a role in the regulation of carnobacteriocin B2. Transcription analysis indicated that the operons cbnXY, cbnB2-cbiB2, and cbnBM1-cbiBM1 (with the latter encoding carnobacteriocin BM1 and its immunity protein on the chromosome) and two small transcripts containing cbnS are transcribed only in induced cultures. These transcripts are coregulated and subject to inducer-mediated transcriptional control. Similar regulation of the cbn operons is mirrored by the similarity in the nucleotide sequence of their promoter regions, all of which contain two imperfect direct repeats resembling those in Agr-like regulated promoters upstream of the transcription start sites.

Amino Acid Sequence↗

PubMed1996

Involvement of stress protein PspA (phage shock protein A) of Escherichia coli in maintenance of the protonmotive force under stress conditions.

The expression of specific PhoE mutant proteins leads to induction of the expression of the psp operon of Escherichia coli and the export of various plasmid-encoded precursors is retarded in a pspA mutant strain. Here, we have investigated the specific role of various Psp proteins in the export process. PspB and PspC are both inner membrane proteins that are involved in the regulation of the transcription of the psp operon. Precursor PhoE translocation was retarded in a pspB mutant strain to a similar extent as in a pspA mutant strain. The reduced translocation efficiencies in the various psp mutants could be complemented by expression of PspA from a plasmid, indicating that only PspA is required for efficient translocation. Mutant prePhoE proteins that can be translocated independently of the deltamu H+ appeared to translocate equally efficiently in a wild-type and in a pspA mutant strain. Furthermore, quantitative in vivo determination of the deltamu H+ showed that it specifically decreased in a pspA mutant strain upon expression of plasmid-encoded (mutant) prePhoE protein. Apparently, the translocation defects observed in a psp mutant strain are caused by a decrease of the delta mu H+ and PspA functions by maintaining the delta mu H+ under these conditions.

Bacterial Proteins↗

PubMed1996

The qmeA (ts) mutation of Escherichia coli is localized in the fabI gene, which encodes enoyl-ACP reductase.

The phenotypes of temperature-sensitive qmeA and fabI mutants of Escherichia coli appear to be very similar. Furthermore, the qmeA mutation could be complemented by the fabI gene on a plasmid, and the fabI allele derived from the qmeA mutant strain harbours a nucleotide substitution identical to that from a previously characterized fabI mutant. These results show that the qmeA gene is, in fact, identical to the fabI gene, which encodes enoyl-ACP reductase, involved in fatty acid elongation.

Enoyl-(Acyl-Carrier Protein) Reductase (NADPH, B-S↗

PubMed1995

Characterization of an Escherichia coli rotA mutant, affected in periplasmic peptidyl-prolyl cis/trans isomerase.

The rotA gene of Escherichia coli encodes a peptidyl-prolyl cis/trans isomerase (PPIase), which is supposed to catalyse protein folding in the periplasm. To investigate the importance of the enzyme, the rotA gene was cloned and a chromosomal deletion mutant was created. The rotA mutant was normally viable. No residual PPIase activity could be detected in the periplasmic fraction of the mutant. Comparison of the patterns of periplasmic and outer membrane proteins by SDS-PAGE revealed no differences in protein composition between the rotA mutant and its parental strain. Similarly, the kinetics of periplasmic protein folding and outer membrane protein assembly appeared unaffected by the rotA mutation. Our results show that the periplasmic PPIase of E. coli is not essential and that the protein does not play an important role in protein folding.

Amino Acid Isomerases↗

PubMed1993

Expression of the pspA gene stimulates efficient protein export in Escherichia coli.

Expression of several mutant forms of outer membrane protein PhoE of Escherichia coli, which are disturbed in normal biogenesis, resulted in high expression of a 26 kDa protein. This 26 kDa protein fractionated as a peripherally bound inner membrane protein. It appeared to be identical to a previously identified protein (PspA = phage shock protein A) of unknown function that is induced upon infection of E. coli with filamentous phages. PspA was not expressed upon synthesis of mutant PhoE proteins in a secB mutant, nor upon expression of a PhoE mutant that lacks the signal sequence, suggesting that entrance into the export pathway of prePhoE is essential for induction. PspA synthesis was also induced under other conditions that are known to block the export apparatus, i.e. in secA, secD and secF mutants when grown at their non-permissive temperature or upon induction of the synthesis of MalE-LacZ or LamB-LacZ hybrid proteins. The inducing conditions for PspA synthesis suggested a role for this protein in export. In vivo pulse-chase experiments showed that the translocation of (mutant) prePhoE and of the precursors of other exported proteins was retarded in a pspA mutant strain. Also, in in vitro translocation assays, a role for PspA in protein transport could be demonstrated.

Bacterial Outer Membrane Proteins↗

PubMedDate not supplied

Exopolysaccharides produced by Lactococcus lactis: from genetic engineering to improved rheological properties?

Over the last years, important advances have been made in the study of the production of exopolysaccharides (EPS) by several lactic acid bacteria, including Lactococcus lactis. From different EPS-producing lactococcal strains the specific eps gene clusters have been characterised. They contain eps genes, which are involved in EPS repeating unit synthesis, export, polymerisation, and chain length determination. The function of the glycosyltransferase genes has been established and the availability of these genes opened the way to EPS engineering. In addition to the eps genes, biosynthesis of EPS requires a number of housekeeping genes that are involved in the metabolic pathways leading to the EPS-building blocks, the nucleotide sugars. The identification and characterisation of several of these housekeeping genes (galE, galU, rfbABCD) allows the design of metabolic engineering strategies that should lead to increased EPS production levels by L. lactis. Finally, model development has been initiated in order to predict the physicochemical consequences of the addition of a EPS to a product.

Genes, Bacterial↗