Biomedical subjects

Jan Kok

Publications and source records attributed to Jan Kok.

At least 19 recordsLinked to original sources

PubMed2007

Transcriptome analysis of temporal regulation of carbon metabolism by CcpA in Bacillus subtilis reveals additional target genes.

The pleiotropic regulator of carbon metabolism in Gram-positive bacteria, CcpA, regulates gene expression by binding to so-called cre elements, which are located either upstream or in promoter regions, or in open-reading frames. In this study we compared the transcriptomes of Bacillus subtilis 168 and its ccpA deletion mutant during growth in glucose-containing rich medium. Although growth was similar, glucose was completely consumed by the wild-type strain in the stationary phase, while it was still present in the culture of the mutant. At that stage, direct and indirect effects on gene expression were observed. During exponential growth, CcpA mainly influences the carbohydrate and energy metabolism, whereas from transition phase onwards its function expands on a broader range of physiological processes including nucleotide metabolism, cell motility and protein synthesis. A genome wide search revealednew putative cre sites, which could function in vivo according to our transcriptome data. Comparison of our data with published transcriptome data of ccpA mutant analysis in the exponential growth phase confirmed earlier identified CcpA regulon members. It also allowed identification of potential new CcpA-repressed genes, amongst others ycgN and the ydh operon. Novel activated members include opuE andthe opuAABC, yhb and man operons, which all have a putative cre site that appears to be dependent on helical topology. A comparative analysis of these genes with the known activated genes i.e.ackA and pta revealed the presence of a possible upstream activating region (UAR) as has been shown to be functional for the activation of ackA. The data suggest that at later growth phases CcpA may regulate gene expression by itself or complexed with other, yet unknown cofactors.

Bacillus subtilis↗

PubMed2006

Time-resolved determination of the CcpA regulon of Lactococcus lactis subsp. cremoris MG1363.

Carbon catabolite control protein A (CcpA) is the main regulator involved in carbon catabolite repression in gram-positive bacteria. Time series gene expression analyses of Lactococcus lactis MG1363 and L. lactis MG1363DeltaccpA using DNA microarrays were used to define the CcpA regulon of L. lactis. Based on a comparison of the transcriptome data with putative CcpA binding motifs (cre sites) in promoter sequences in the genome of L. lactis, 82 direct targets of CcpA were predicted. The main differences in time-dependent expression of CcpA-regulated genes were differences between the exponential and transition growth phases. Large effects were observed for carbon and nitrogen metabolic genes in the exponential growth phase. Effects on nucleotide metabolism genes were observed primarily in the transition phase. Analysis of the positions of putative cre sites revealed that there is a link between either repression or activation and the location of the cre site within the promoter region. Activation was observed when putative cre sites were located upstream of the hexameric -35 sequence at an average position of -56.5 or further upstream with decrements of 10.5 bp. Repression was observed when the cre site was located in or downstream of putative -35 and -10 sequences. The highest level of repression was observed when the cre site was present at a defined side of the DNA helix relative to the canonical -10 sequence. Gel retardation experiments, Northern blotting, and enzyme assays showed that CcpA represses its own expression and activates the expression of the divergently oriented prolidase-encoding pepQ gene, which constitutes a link between regulation of carbon metabolism and regulation of nitrogen metabolism.

Bacterial Proteins↗

PubMed2006

The alpha-phosphoglucomutase of Lactococcus lactis is unrelated to the alpha-D-phosphohexomutase superfamily and is encoded by the essential gene pgmH.

alpha-Phosphoglucomutase (alpha-PGM) plays an important role in carbohydrate metabolism by catalyzing the reversible conversion of alpha-glucose 1-phosphate to glucose 6-phosphate. Isolation of alpha-PGM activity from cell extracts of Lactococcus lactis strain MG1363 led to the conclusion that this activity is encoded by yfgH, herein renamed pgmH. Its gene product has no sequence homology to proteins in the alpha-d-phosphohexomutase superfamily and is instead related to the eukaryotic phosphomannomutases within the haloacid dehalogenase superfamily. In contrast to known bacterial alpha-PGMs, this 28-kDa enzyme is highly specific for alpha-glucose 1-phosphate and glucose 6-phosphate and showed no activity for mannose phosphate. To elucidate the function of pgmH, the metabolism of glucose and galactose was characterized in mutants overproducing or with a deficiency of alpha-PGM activity. Overproduction of alpha-PGM led to increased glycolytic flux and growth rate on galactose. Despite several attempts, we failed to obtain a deletion mutant of pgmH. The essentiality of this gene was proven by using a conditional knock-out strain in which a native copy of the gene was provided in trans under the control of the nisin promoter. Growth of this strain was severely impaired when alpha-PGM activity was below the control level. We show that the novel L. lactis alpha-PGM is the only enzyme that mediates the interconversion of alpha-glucose 1-phosphate to glucose 6-phosphate and is essential for growth.

Amino Acid Sequence↗

PubMed2006

BAGEL: a web-based bacteriocin genome mining tool.

A common problem in the annotation of open reading frames (ORFs) is the identification of genes that are functionally similar but have limited or no sequence homology. This is particularly the case for bacteriocins, a very diverse group of antimicrobial peptides produced by bacteria and usually encoded by small, poorly conserved ORFs. ORFs surrounding bacteriocin genes are often biosynthetic genes. This information can be used to locate putative structural bacteriocin genes. Here, we describe BAGEL, a web server that identifies putative bacteriocin ORFs in a DNA sequence using novel, knowledge-based bacteriocin databases and motif databases. Many bacteriocins are encoded by small genes that are often omitted in the annotation process of bacterial genomes. Thus, we have implemented ORF detection using a number of published ORF prediction tools. In addition, BAGEL takes into account the genomic context, i.e. for each potential bacteriocin-encoding ORF, the sequence of the surrounding region on the genome is analyzed for genes that might encode proteins involved in biosynthesis, transport, regulation and/or immunity. These innovations make BAGEL unique in its ability to detect putative bacteriocin gene clusters in (new) bacterial genomes. BAGEL is freely accessible at: http://bioinformatics.biol.rug.nl/websoftware/bagel.

Amino Acid Motifs↗

PubMed2006

Regulation of glutamine and glutamate metabolism by GlnR and GlnA in Streptococcus pneumoniae.

Several genes involved in nitrogen metabolism are known to contribute to the virulence of pathogenic bacteria. Here, we studied the function of the nitrogen regulatory protein GlnR in the Gram-positive human pathogen Streptococcus pneumoniae. We demonstrate that GlnR mediates transcriptional repression of genes involved in glutamine synthesis and uptake (glnA and glnPQ), glutamate synthesis (gdhA), and the gene encoding the pentose phosphate pathway enzyme Zwf, which forms an operon with glnPQ. Moreover, the expression of gdhA is also repressed by the pleiotropic regulator CodY. The GlnR-dependent regulation occurs through a conserved operator sequence and is responsive to the concentration of glutamate, glutamine, and ammonium in the growth medium. By means of in vitro binding studies and transcriptional analyses, we show that the regulatory function of GlnR is dependent on GlnA. Mutants of glnA and glnP displayed significantly reduced adhesion to Detroit 562 human pharyngeal epithelial cells, suggesting a role for these genes in the colonization of the host by S. pneumoniae. Thus, our results provide a thorough insight into the regulation of glutamine and glutamate metabolism of S. pneumoniae mediated by both GlnR and GlnA.

Base Sequence↗

PubMed2006

Natural sweetening of food products by engineering Lactococcus lactis for glucose production.

We show that sweetening of food products by natural fermentation can be achieved by a combined metabolic engineering and transcriptome analysis approach. A Lactococcus lactis ssp. cremoris strain was constructed in which glucose metabolism was completely disrupted by deletion of the genes coding for glucokinase (glk), EII(man/glc) (ptnABCD), and the newly discovered glucose-PTS EII(cel) (ptcBAC). After introducing the lactose metabolic genes, the deletion strain could solely ferment the galactose moiety of lactose, while the glucose moiety accumulated extracellularly. Additionally, less lactose remained in the medium after fermentation. The resulting strain can be used for in situ production of glucose, circumventing the need to add sweeteners as additional ingredients to dairy products. Moreover, the enhanced removal of lactose achieved by this strain could be very useful in the manufacture of products for lactose intolerant individuals.

Escherichia coli Proteins↗

PubMed2006

SIMAGE: simulation of DNA-microarray gene expression data.

BACKGROUND: Simulation of DNA-microarray data serves at least three purposes: (i) optimizing the design of an intended DNA microarray experiment, (ii) comparing existing pre-processing and processing methods for best analysis of a given DNA microarray experiment, (iii) educating students, lab-workers and other researchers by making them aware of the many factors influencing DNA microarray experiments. RESULTS: Our model has multiple layers of factors influencing the experiment. The relative influence of such factors can differ significantly between labs, experiments within labs, etc. Therefore, we have added a module to roughly estimate their parameters from a given data set. This guarantees that our simulated data mimics real data as closely as possible. CONCLUSION: We introduce a model for the simulation of dual-dye cDNA-microarray data closely resembling real data and coin the model and its software implementation "SIMAGE" which stands for simulation of microarray gene expression data. The software is freely accessible at: http://bioinformatics.biol.rug.nl/websoftware/simage.

Algorithms↗

PubMed2006

High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis.

Enterocin P (EntP), a sec-dependent bacteriocin from Enterococcus faecium P13, was produced by Lactococcus lactis. The EntP structural gene (entP) with or without the EntP immunity gene (entiP) was cloned in (1), plasmid pMG36c under control of the lactococcal constitutive promoter P32, (2) in plasmid pNG8048e under control of the inducible PnisA promoter, and (3) in the integration vector pINT29. Introduction of the recombinant vectors in L. lactis resulted in production of biologically active EntP in the supernatants of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000, and the coproduction of nisin A and EntP in L. lactis subsp. lactis DPC5598. The level of production of EntP, detected and quantified by specific anti-EntP antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, by the recombinant L. lactis strains depended on the host strain, the expression vector, and the presence of the entiP gene in the constructs of the recombinant L. lactis strains. The highest amount of EntP was produced with derivatives containing entP and entiP, for both L. lactis IL1403 and L. lactis NZ9000. These derivatives produced up to five- to six-fold more EntP than E. faecium P13. Mass spectrometry analysis revealed that EntP purified from L. lactis IL1403 (pJP214) has a molecular mass identical to that purified from E. faecium P13, suggesting that the synthesis, processing, and secretion of EntP progresses efficiently in recombinant L. lactis hosts.

Bacterial Proteins↗

PubMed2006

Characterization of the lytic-lysogenic switch of the lactococcal bacteriophage Tuc2009.

Tuc2009 is a temperate bacteriophage of Lactococcus lactis subsp. cremoris UC509 which encodes a CI- and Cro-type lysogenic-lytic switch region. A helix-swap of the alpha3 helices of the closely related CI-type proteins from the lactococcal phages r1t and Tuc2009 revealed the crucial elements involved in DNA recognition while also pointing to conserved functional properties of phage CI proteins infecting different hosts. CI-type proteins have been shown to bind to specific sequences located in the intergenic switch region, but to date, no detailed binding studies have been performed on lactococcal Cro analogues. Experiments shown here demonstrate alternative binding sites for these two proteins of Tuc2009. CI2009 binds to three inverted repeats, two within the intergenic region and one within the cro2009 gene. This DNA-binding pattern appears to be conserved among repressors of lactococcal and streptococcal phages. The Cro2009 protein appears to bind to three direct repeats within the intergenic region causing distortion of the bound DNA.

DNA, Viral↗

PubMed2006

Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria.

Mucosal immunization with subunit vaccines requires new types of antigen delivery vehicles and adjuvants for optimal immune responses. We have developed a non-living and non-genetically modified gram-positive bacterial delivery particle (GEM) that has built-in adjuvant activity and a high loading capacity for externally added heterologous antigens that are fused to a high affinity binding domain. This binding domain, the protein anchor (PA), is derived from the Lactococcus lactis AcmA cell-wall hydrolase, and contains three repeats of a LysM-type cell-wall binding motif. Antigens are produced as antigen-PA fusions by recombinant expression systems that secrete the hybrid proteins into the culture growth medium. GEM particles are then used as affinity beads to isolate the antigen-PA fusions from the complex growth media in a one step procedure after removal of the recombinant producer cells. This procedure is also highly suitable for making multivalent vaccines. The resulting vaccines are stable at room temperature, lack recombinant DNA, and mimic pathogens by their bacterial size, surface display of antigens and adjuvant activity of the bacterial components in the GEM particles. The GEM-based vaccines do not require additional adjuvant for eliciting high levels of specific antibodies in mucosal and systemic compartments.

Adjuvants, Immunologic↗

PubMed2006

To have neighbour's fare: extending the molecular toolbox for Streptococcus pneumoniae.

In past years, several useful genetic tools have been developed to study the molecular biology of Streptococcus pneumoniae. In order to extend the existing spectrum of tools, advantage was taken of the toolbox originally developed for the closely related bacterium Lactococcus lactis, which was adapted for the manipulation of S. pneumoniae. The modified tools are as follows. (i) An improved nisin-inducible (over)expression system (NICE). The nisRK genes, encoding a two-component system essential for transcriptional activation in response to nisin, were integrated into the bgaA locus of S. pneumoniae D39. In this strain, D39nisRK, addition of nisin resulted in the overexpression of several genes placed under the control of the nisin-inducible promoter, while no detectable expression was observed in the absence of nisin. (ii) A lacZ reporter system. Using strain D39nisRK, which lacks endogenous beta-galactosidase activity, the usefulness of the lacZ reporter vector pORI13 for the generation of chromosomal transcriptional fusions was demonstrated. In addition, the repA gene, necessary for the replication of pORI13, was introduced into the bgaA locus, thereby generating a background for plasmid-based promoter expression studies. (iii) A simplified chemically defined medium, which supports growth of all sequenced S. pneumoniae strains to a level comparable to that in complex medium. (iv) A system for the introduction of unmarked deletions and mutations into the chromosome, which is independent of the genotype of the target strain. Most of these systems were successfully applied in strains R6 and TIGR4 as well. In addition, the tools offer several improvements and advantages compared to existing ones. Thus, the molecular toolbox for S. pneumoniae has been successfully extended.

Biotechnology↗

PubMed2006

Different subcellular locations of secretome components of Gram-positive bacteria.

Gram-positive bacteria contain different types of secretion systems for the transport of proteins into or across the cytoplasmic membrane. Recent studies on subcellular localization of specific components of these secretion systems and their substrates have shown that they can be present at various locations in the cell. The translocons of the general Sec secretion system in the rod-shaped bacterium Bacillus subtilis have been shown to localize in spirals along the cytoplasmic membrane, whereas the translocons in the coccoid Streptococcus pyogenes are located in a microdomain near the septum. In both bacteria the Sec translocons appear to be located near the sites of cell wall synthesis. The Tat secretion system, which is used for the transport of folded proteins, probably localizes in the cytoplasmic membrane and at the cell poles of B. subtilis. In Lactococcus lactis the ABC transporter dedicated to the transport of a small antimicrobial peptide is distributed throughout the membrane. Possible mechanisms for maintaining the localization of these secretion machineries involve their interaction with proteins of the cytoskeleton or components of the cell wall synthesis machinery, or the presence of lipid subdomains surrounding the transport systems.

Bacterial Proteins↗

PubMed2006

LmrCD is a major multidrug resistance transporter in Lactococcus lactis.

When Lactococcus lactis is challenged with drugs it displays a multidrug resistance (MDR) phenotype. In silico analysis of the genome of L. lactis indicates the presence of at least 40 putative MDR transporters, of which only four, i.e. the ABC transporters LmrA, LmrC and LmrD, and the major facilitator LmrP, have been experimentally associated with the MDR. To understand the molecular basis of the MDR phenotype in L. lactis, we have performed a global transcriptome analysis comparing four independently isolated drug-resistant strains of L. lactis with the wild-type strain. The results show a strong and consistent upregulation of the lmrC and lmrD genes in all four strains, while the mRNA levels of other putative MDR transporters were not significantly altered. Deletion of lmrCD renders L. lactis sensitive to several toxic compounds, and this phenotype is associated with a reduced ability to secrete these compounds. Another gene, which is strongly upregulated in all mutant strains, specifies LmrR (YdaF), a local transcriptional repressor of lmrCD that belongs to the PadR family of transcriptional regulators and that binds to the promoter region of lmrCD. These results demonstrate that the heterodimeric MDR ABC transporter LmrCD is a major determinant of both acquired and intrinsic drug resistance of L. lactis.

ATP-Binding Cassette Transporters↗

PubMed2006

Transcriptome analysis reveals mechanisms by which Lactococcus lactis acquires nisin resistance.

Nisin, a posttranslationally modified antimicrobial peptide produced by Lactococcus lactis, is widely used as a food preservative. Yet, the mechanisms leading to the development of nisin resistance in bacteria are poorly understood. We used whole-genome DNA microarrays of L. lactis IL1403 to identify the factors underlying acquired nisin resistance mechanisms. The transcriptomes of L. lactis IL1403 and L. lactis IL1403 Nis(r), which reached a 75-fold higher nisin resistance level, were compared. Differential expression was observed in genes encoding proteins that are involved in cell wall biosynthesis, energy metabolism, fatty acid and phospholipid metabolism, regulatory functions, and metal and/or peptide transport and binding. These results were further substantiated by showing that several knockout and overexpression mutants of these genes had strongly altered nisin resistance levels and that some knockout strains could no longer become resistant to the same level of nisin as that of the wild-type strain. The acquired nisin resistance mechanism in L. lactis is complex, involving various different mechanisms. The four major mechanisms are (i) preventing nisin from reaching the cytoplasmic membrane, (ii) reducing the acidity of the extracellular medium, thereby stimulating the binding of nisin to the cell wall, (iii) preventing the insertion of nisin into the membrane, and (iv) possibly transporting nisin across the membrane or extruding nisin out of the membrane.

Anti-Bacterial Agents↗

PubMed2006

Novel surface display system for proteins on non-genetically modified gram-positive bacteria.

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.

Administration, Intranasal↗

PubMed2006

GlnR-mediated regulation of nitrogen metabolism in Lactococcus lactis.

We show that the nitrogen regulatory protein GlnR of Lactococcus lactis represses transcription of the amtB-glnK, glnRA, and glnPQ operons. This likely occurs through a conserved DNA motif, 5'-TGTNA-7N-TNACAT-3', and takes place in response to extracellular glutamine and ammonium. GlnR-independent repression of amtB-glnK is mediated by the pleiotropic nitrogen regulator CodY.

Bacterial Proteins↗

PubMed2006

Identification and functional characterization of the Lactococcus lactis CodY-regulated branched-chain amino acid permease BcaP (CtrA).

Transcriptome analyses have previously revealed that a gene encoding the putative amino acid transporter CtrA (YhdG) is one of the major targets of the pleiotropic regulator CodY in Lactococcus lactis and Bacillus subtilis. The role of ctrA in L. lactis was further investigated with respect to both transport activity as well as CodY-mediated regulation. CtrA is required for optimal growth in media containing free amino acids as the only amino acid source. Amino acid transport studies showed that ctrA encodes a secondary amino acid transport system that is specific for branched-chain amino acids (BCAAs) (isoleucine, leucine, and valine) and methionine, which is in disagreement with its previously proposed function (a cationic amino acid transporter), which was assigned based on homology. We propose to rename CtrA BcaP, for branched-chain amino acid permease. BcaP is a member of a group of conserved transport systems, as homologs are widely distributed among gram-positive bacteria. Deletion of bcaP resulted in the loss of most of the BCAA uptake activity of L. lactis, indicating that BcaP is the major BCAA carrier of this organism. Deletion of bcaP together with a second (putative) BCAA permease, encoded by brnQ, further reduced the viability of the strain. DNA microarray analysis showed that deletion of bcaP predominantly affects genes belonging to the regulons of the transcriptional regulator CodY, which is involved in global nitrogen metabolism and needs BCAAs for its activation, and of CmbR, which is involved in sulfur amino acid metabolism.

Amino Acid Transport Systems↗

PubMed2005

The Lactococcus lactis CodY regulon: identification of a conserved cis-regulatory element.

CodY of Lactococcus lactis MG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. DNA microarray analysis, comparing the expression profiles of L. lactis MG1363 and an isogenic strain in which codY was mutated, was used to determine the CodY regulon. In peptide-rich medium and exponentially growing cells, where CodY exerts strong repressing activity, the expression of over 30 genes was significantly increased upon removal of codY. The differentially expressed genes included those predominantly involved in amino acid transport and metabolism. In addition, several genes belonging to other functional categories were derepressed, stressing the pleiotropic role of CodY. Scrutinizing the transcriptome data with bioinformatics tools revealed the presence of a novel over-represented motif in the upstream regions of several of the genes derepressed in L. lactis MG1363DeltacodY. Evidence is presented that this 15-bp cis-sequence, AATTTTCWGAAAATT, serves as a high affinity binding site for CodY, as shown by electrophoretic mobility shift assays and DNase I footprinting analyses. The presence of this CodY-box is sufficient to evoke CodY-mediated regulation in vivo. A copy of this motif is also present in the upstream region of codY itself. It is shown that CodY regulates its own synthesis and requires the CodY-box and branched-chain amino acids to interact with its promoter.

Bacterial Proteins↗