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O P Kuipers

Publications and source records attributed to O P Kuipers.

At least 19 recordsLinked to original sources
PubMed2006

Single cell analysis of gene expression patterns of competence development and initiation of sporulation in Bacillus subtilis grown on chemically defined media.

AIM: Understanding the basis for the heterogeneous (or bistable) expression patterns of competence development and sporulation in Bacillus subtilis. METHODS AND RESULTS: Using flow cytometric analyses of various promoter-GFP fusions, we have determined the single-cell gene expression patterns of competence development and initiation of sporulation in a chemically defined medium (CDM) and in biofilms. CONCLUSIONS: We show that competence development and initiation of sporulation in a CDM are still initiated in a bistable manner, as is the case in complex media, but are sequential in their timing. Furthermore, we provide experimental proof that competence and sporulation can develop under conditions that normally do not trigger these processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Some pathogens are able to develop natural competence, which is a serious medical problem with the increased acquired multi-drug resistance of these organisms. Another adaptive microbial response is spore formation. Because of their heat resistance and hydrophobicity, spores of a variety of species are of major concern for the food industry. Using the model organism B. subtilis, we show that competence development and sporulation are initiated in a bistable and sequential manner. We furthermore show that both processes may be noise-based, which has major implications for the control of unwanted differentiation processes in pathogenic and food-spoilage micro-organisms.

Bacillus subtilis↗
PubMed2004

Engineering of quorum-sensing systems for improved production of alkaline protease by Bacillus subtilis.

AIM: Engineering of Rap-Phr quorum-sensing systems of Bacillus subtilis and subsequent evaluation of the transcription of the aprE gene, encoding a major extracellular alkaline protease. METHODS AND RESULTS: Addition of synthetic Phr pentapeptides to the growth medium, or overproduction of pre-Phr peptides, slightly improved the transcription of the aprE gene in B. subtilis. Disruption of certain rap genes similarly improved the transcription of the aprE gene. The production of extracellular proteolytic enzymes was increased when the rapA mutation was combined with a degU32 (Hy) mutation for hyper-secretion. CONCLUSIONS: Certain Rap-Phr systems of B. subtilis seem to suppress extracellular AprE production. Although this may be an important feature under natural conditions, repression of AprE production by these systems is not desirable under fermentation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the levels of aprE transcriptional increase in this study are moderate, engineering of Rap-Phr systems may be used to improve the yield of Bacillus strains that are used for the production of the extracellular protease AprE, or Bacillus strains that use of the aprE promoter for the production of a heterologous protein.

Bacillus subtilis↗
PubMed2004

The Bacillus secretion stress response is an indicator for alpha-amylase production levels.

AIMS: Overproduced alpha-amylases in Bacillus subtilis provoke a specific stress response involving the CssRS two-component system, which controls expression of the HtrA and HtrB proteases. Previously, the B. subtilis TepA protein was implicated in high-level alpha-amylase secretion. Our present studies were aimed at investigating a possible role of TepA in secretion stress management, and characterizing the intensity of the secretion stress response in relation to alpha-amylase production. METHODS AND RESULTS: The expression of a transcriptional htrB-lacZ gene fusion, and the levels of alpha-amylase production were monitored simultaneously using tepA mutant B. subtilis strains. TepA was shown to be dispensable for secretion stress management. Importantly, however, the levels of htrB-lacZ expression can be correlated with the levels of alpha-amylase production. CONCLUSION: Our observations show that the secretion stress response can serve as an indicator for alpha-amylase production levels. SIGNIFICANCE AND IMPACT OF STUDY: Conceivably, this stress response can be employed to monitor the biotechnological production of various secretory proteins by the Bacillus cell factory.

Acyltransferases↗
PubMed2004

Mechanism of transcription activation at the comG promoter by the competence transcription factor ComK of Bacillus subtilis.

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type alpha-subunits and with C-terminal deletion mutants of the alpha-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.

Bacillus subtilis↗
PubMed2001

Casitone-mediated expression of the prtP and prtM genes in Lactococcus lactis subsp. lactis BGIS29.

Casitone added to chemically defined medium (CDM) specifically influenced the regulation of the proteinase activity in the natural isolate Lactococcus lactis subsp. lactis BGIS29. Comparative analysis of the influence of casitone present in CDM on the proteolytic activity of strain BGIS29 and of L. lactis subsp. cremoris strains SK11 and Wg2 indicated that the proteolytic activity of strains BGIS29 and SK11 is casitone-dependent, whereas that of strain Wg2 is not. The regulatory region of the prt genes of strain BGIS29 was cloned and sequenced. The nucleotide sequence of the prt regulatory region of strain BGIS29 was distinctly different from that of L. lactis subsp. cremoris strains SK11 and Wg2. Transcriptional gene fusions with the Escherichia coli beta-glucuronidase gene ( gusA) were used to study medium-dependent expression of two divergent prtP and prtM promoters of strain BGIS29 (designated P (prtP) and P (prtM), respectively). beta-Glucuronidase assays and Northern blot analysis showed that the activities of P (prtP) and P (prtM) are controlled by casitone at the transcriptional level.

Amino Acid Sequence↗
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

Cold shock proteins of Lactococcus lactis MG1363 are involved in cryoprotection and in the production of cold-induced proteins.

Members of the group of 7-kDa cold-shock proteins (CSPs) are the proteins with the highest level of induction upon cold shock in the lactic acid bacterium Lactococcus lactis MG1363. By using double-crossover recombination, two L. lactis strains were generated in which genes encoding CSPs are disrupted: L. lactis NZ9000 Delta AB lacks the tandemly orientated cspA and cspB genes, and NZ9000 Delta ABE lacks cspA, cspB, and cspE. Both strains showed no differences in growth at normal and at low temperatures compared to that of the wild-type strain, L. lactis NZ9000. Two-dimensional gel electrophoresis showed that upon disruption of the cspAB genes, the production of remaining CspE at low temperature increased, and upon disruption of cspA, cspB, and cspE, the production of CspD at normal growth temperatures increased. Northern blot analysis showed that control is most likely at the transcriptional level. Furthermore, it was established by a proteomics approach that some (non-7-kDa) cold-induced proteins (CIPs) are not cold induced in the csp-lacking strains, among others the histon-like protein HslA and the signal transduction protein LlrC. This supports earlier observations (J. A. Wouters, M. Mailhes, F. M. Rombouts, W. M. De Vos, O. P. Kuipers, and T. Abee, Appl. Environ. Microbiol. 66:3756-3763, 2000). that the CSPs of L. lactis might be directly involved in the production of some CIPs upon low-temperature exposure. Remarkably, the adaptive response to freezing by prior exposure to 10 degrees C was significantly reduced in strain NZ9000 Delta ABE but not in strain NZ9000 Delta AB compared to results with wild-type strain NZ9000, indicating a notable involvement of CspE in cryoprotection.

Adaptation, Physiological↗
PubMed2001

Regulatory functions of serine-46-phosphorylated HPr in Lactococcus lactis.

In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a beta-glucoside-specific EII and a 6-P-beta-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methyl beta-D-thiogalactoside (TMG) and 2-deoxy-D-glucose (2-DG). In vivo experiments with the ptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

Bacterial Proteins↗
PubMed2001

Activation of silent gal genes in the lac-gal regulon of Streptococcus thermophilus.

Streptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with Escherichia coli demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the order galKTE, which was preceded by a divergently transcribed regulator gene, galR, and followed by a galM gene and the lactose operon lacSZ. Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the galKTE genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal(+) mutant NZ302G, the transcription of the galR gene was induced by growth on lactose. Disruption of galR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression. Sequence analysis of the gal promoter regions of NZ302G and nine other independently isolated Gal(+) mutants of CNRZ 302 revealed mutations at three positions in the galK promoter region, which included substitutions at positions -9 and -15 as well as a single-base-pair insertion at position -37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis of gusA reporter gene fusions in strains containing the mutated promoters suggested that they were gal promoter-up mutations. We propose that poor expression of the gal genes in the galactose-negative S. thermophilus CNRZ 302 is caused by naturally occurring mutations in the galK promoter.

Base Sequence↗
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

Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity.

Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation. Here we use genetically engineered nisin variants to identify the structural requirements for the interaction of the peptide with lipid II. Mutations affecting the conformation of the N-terminal part of nisin comprising rings A through C, e.g. [S3T]nisin, led to reduced binding and increased the peptide concentration necessary for pore formation. The binding constant for the S3T mutant was 0.043 x 10(7) m(-1) compared with 2 x 10(7) m(-1) for the wild-type peptide, and the minimum concentration for pore formation increased from the 1 nm to the 50 nm range. In contrast, peptides mutated in the flexible hinge region, e.g. [DeltaN20/DeltaM21]nisin, were completely inactive in the pore formation assay, but were reduced to some extent in their in vivo activity. We found the remaining in vivo activity to result from the unaltered capacity of the mutated peptide to bind to lipid II and thus to inhibit its incorporation into the peptidoglycan network. Therefore, through interaction with the membrane-bound cell wall precursor lipid II, nisin inhibits peptidoglycan synthesis and forms highly specific pores. The combination of two killing mechanisms in one molecule potentiates antibiotic activity and results in nanomolar MIC values, a strategy that may well be worth considering for the construction of novel antibiotics.

Anti-Bacterial Agents↗
PubMed2000

The role of cold-shock proteins in low-temperature adaptation of food-related bacteria.

There is a considerable interest in the cold adaptation of food-related bacteria, including starter cultures for industrial food fermentations, food spoilage bacteria and food-borne pathogens. Mechanisms that permit low-temperature growth involve cellular modifications for maintaining membrane fluidity, the uptake or synthesis of compatible solutes, the maintenance of the structural integrity of macromolecules and macromolecule assemblies, such as ribosomes and other components that affect gene expression. A specific cold response that is shared by nearly all food-related bacteria is the induction of the synthesis so-called cold-shock proteins (CSPs), which are small (7 kDa) proteins that are involved in mRNA folding, protein synthesis and/or freeze protection. In addition, CSPs are able to bind RNA and it is believed that these proteins act as RNA chaperones, thereby reducing the increased secondary folding of RNA at low temperatures. In this review established and novel aspects concerning the structure, function and control of these CSPs are discussed. A model for bacterial cold adaptation, with a central role for ribosomal functioning, and possible mechanisms for low-temperature sensing are discussed.

Adaptation, Biological↗
PubMed2000

Current strategies for improving food bacteria.

Novel concepts and methodologies are emerging that hold great promise for the directed improvement of food-related bacteria, specifically lactic acid bacteria. Also, the battle against food spoilage and pathogenic bacteria can now be fought more effectively. Here we describe recent advances in microbial physiology and genomic research of these organisms that enable novel strategies for obtaining safe, healthy, and good-tasting fermented food products.

Bacteriocins↗
PubMed2000

Lactic acid bacteria: the bugs of the new millennium.

Lactic acid bacteria (LABs) are widely used in the manufacturing of fermented food and are among the best-studied microorganisms. Detailed knowledge of a number of physiological traits has opened new potential applications for these organisms in the food industry, while other traits might be beneficial for human health. Important new developments have been made in the research of LABs in the areas of multidrug resistance, bacteriocins and quorum sensing, osmoregulation, proteolysis, autolysins and bacteriophages. Recently, progress has been made in the construction of food-grade genetically modified LABs.

Bacteriocins↗
PubMed2000

Engineering a disulfide bond and free thiols in the lantibiotic nisin Z.

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.

Amino Acid Sequence↗
PubMed2000

Molecular and functional analyses of the metC gene of Lactococcus lactis, encoding cystathionine beta-lyase.

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine beta-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an alpha, gamma elimination. With methionine as a substrate, it produces volatile sulfur compounds which are important for flavor formation in Gouda cheese. The metC gene, which encodes CBL, was cloned from the Lactococcus lactis model strain MG1363 and from strain B78, isolated from a cheese starter culture and known to have a high capacity to produce volatile compounds. The metC gene was found to be cotranscribed with a downstream cysK gene, which encodes a putative cysteine synthase. The MetC proteins of both strains were overproduced in strain MG1363 with the NICE (nisin-controlled expression) system, resulting in a >25-fold increase in cystathionine lyase activity. A disruption of the metC gene was achieved in strain MG1363. Determination of enzymatic activities in the overproducing and knockout strains revealed that MetC is essential for the degradation of cystathionine but that at least one lyase other than CBL contributes to methionine degradation via alpha, gamma elimination to form volatile aroma compounds.

Amino Acid Sequence↗
PubMed2000

Cloning, characterization, controlled overexpression, and inactivation of the major tributyrin esterase gene of Lactococcus lactis.

The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene. This confirms that EstA is the main enzyme responsible for esterase activity in L. lactis. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway.

Amino Acid Sequence↗