Biomedical subjects

Jan Kok

Publications and source records attributed to Jan Kok.

At least 19 recordsLinked to original sources

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↗

PubMed2005

Projector 2: contig mapping for efficient gap-closure of prokaryotic genome sequence assemblies.

With genome sequencing efforts increasing exponentially, valuable information accumulates on genomic content of the various organisms sequenced. Projector 2 uses (un)finished genomic sequences of an organism as a template to infer linkage information for a genome sequence assembly of a related organism being sequenced. The remaining gaps between contigs for which no linkage information is present can subsequently be closed with direct PCR strategies. Compared with other implementations, Projector 2 has several distinctive features: a user-friendly web interface, automatic removal of repetitive elements (repeat-masking) and automated primer design for gap-closure purposes. Moreover, when using multiple fragments of a template genome, primers for multiplex PCR strategies can also be designed. Primer design takes into account that, in many cases, contig ends contain unreliable DNA sequences and repetitive sequences. Closing the remaining gaps in prokaryotic genome sequence assemblies is thereby made very efficient and virtually effortless. We demonstrate that the use of single or multiple fragments of a template genome (i.e. unfinished genome sequences) in combination with repeat-masking results in mapping success rates close to 100%. The web interface is freely accessible at http://molgen.biol.rug.nl/websoftware/projector2.

Algorithms↗

PubMed2005

A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data.

BACKGROUND: In research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed. RESULTS: A number of validation experiments were performed on Lactococcus lactis IL1403 amplicon-based DNA-microarrays. Using the validation scheme and ANOVA, the factors contributing to the variance in normalized DNA-microarray data were estimated. Day-to-day as well as experimenter-dependent variances were shown to contribute strongly to the variance, while dye and culturing had a relatively modest contribution to the variance. CONCLUSION: Even in cases where 90% of the data were kept for analysis and the experiments were performed under challenging conditions (e.g. on different days), the CV was at an acceptable 25%. Clustering experiments showed that trends can be reliably detected also from genes with very low expression levels. The validation scheme thus allows determining conditions that could be improved to yield even higher DNA-microarray data quality.

Analysis of Variance↗

PubMed2005

Interaction between ArgR and AhrC controls regulation of arginine metabolism in Lactococcus lactis.

The expression of arginine metabolism in Lactococcus lactis is controlled by the two homologous transcriptional regulators ArgR and AhrC. Genome sequence analyses have shown that the occurrence of multiple homologues of the ArgR family of transcriptional regulators is a common feature of many low-G + C Gram-positive bacteria. Detailed studies of ArgR type regulators have previously only been carried out in bacteria containing single regulators. Here, we present a first characterization of the two L. lactis arginine regulators by means of gel retardation and DNase I footprinting. ArgR of L. lactis was shown to bind to the promoter regions of both the arginine biosynthetic argCJDBF operon and the arginine catabolic arcABD1C1C2TD2yvaD operon, but in an arginine-independent manner. Surprisingly, AhrC alone was unable to bind to DNA. Arginine-dependent DNA binding was obtained by mixing the two regulators in gel retardation assays. With both regulators present, the addition of arginine led to increased binding of ArgR-AhrC to the biosynthetic argC promoter but also to diminished binding to the catabolic arcA promoter. Footprinting showed ArgR-AhrC protection of regions containing ARG box operator sequences preceding argC. In the absence of AhrC, ArgR protected sites in the arcA promoter region with similarity to ARG box half-sites, here called ARC boxes. We propose a model for repression of arginine biosynthesis and activation of catabolism by anti-repression, involving arginine-dependent interaction between the two L. lactis regulator proteins, ArgR and AhrC.

Arginine↗

PubMed2005

Comparative and functional genomics of lactococci.

Whole-genome nucleotide sequencing has revolutionized the genetic, biochemical and molecular biology research on bacteria and indeed, many higher organisms. The genome sequences of the strains of two subspecies of Lactococcus lactis, L. lactis subsp. lactis and L. lactis subsp. cremoris, have been determined. These genomic sequences have permitted two important new approaches to be applied in the research of L. lactis. The analysis of the regulation of expression of all genes under specific circumstances at a given point in time is now possible by DNA microarray technology. The elucidation of the full protein complement of the organism as a function of intrinsic or external factors has been made possible by high-throughput protein identification and analysis techniques combined with the gene-derived know-how of the total protein encoding capacity of the genome. These techniques from the genomics arena, transcriptomics and proteomics, have been recently implemented in the study of various aspects of growth and functioning of L. lactis. In this paper we discuss a number of similarities and differences between the two lactococcal genome sequences and review the current status of genomics research in L. lactis. We also propose future directions with respect to both answering fundamental questions more quickly and more completely, as well as opening new avenues for biotechnological applications.

DNA Transposable Elements↗

PubMed2005

Overview on sugar metabolism and its control in Lactococcus lactis - the input from in vivo NMR.

The wide application of lactic acid bacteria in the production of fermented foods depends to a great extent on the unique features of sugar metabolism in these organisms. The relative metabolic simplicity and the availability of genetic tools made Lactococcus lactis the organism of choice to gain insight into metabolic and regulatory networks. In vivo nuclear magnetic resonance has proven a very useful technique to monitor non-invasively the dynamics of intracellular metabolite and co-factor pools following a glucose pulse. Examples of the application of this methodology to identify metabolic bottlenecks and regulatory sites are presented. The use of this information to direct metabolic engineering strategies is illustrated.

Carbohydrate Metabolism↗

PubMed2005

Specificity and selectivity determinants of peptide transport in Lactococcus lactis and other microorganisms.

Peptide transport in microorganisms is important for nutrition of the cell and various signalling processes including regulation of gene expression, sporulation, chemotaxis, competence and virulence development. Peptide transport is mediated via different combinations of ion-linked and ATP-binding cassette (ABC) transporters, the latter utilizing single or multiple peptide-binding proteins with overlapping specificities. The paradigm for research on peptide transport is Lactococcus lactis, in which the uptake of peptides containing essential amino acids is vital for growth on milk proteins. Differential expression and characteristics of peptide-binding proteins in several Lactococcus lactis strains resulted in apparent conflicts with older literature. Recent developments and new data now make the pieces of the puzzle fall back into place again and confirm the view that the oligopeptide-binding proteins determine the uptake selectivity of their cognate ABC transporters. Besides reviewing the current data on binding specificity and transport selectivity of peptide transporters in L. lactis, the possible implications for peptide utilization by other bacterial species are discussed.

ATP-Binding Cassette Transporters↗

PubMed2005

AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning.

AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an N-terminal active site domain and a C-terminal peptidoglycan-binding domain. The active site domain is homologous to that of muramidase-2 of Enterococcus hirae, however, RP-HPLC analysis of muropeptides released from Bacillus subtilis peptidoglycan, after digestion with AcmA, shows that AcmA is an N-acetylglucosaminidase. In the C-terminus of AcmA three highly similar repeated regions of 45 amino acid residues are present, which are separated by short nonhomologous sequences. The repeats of AcmA, which belong to the lysine motif (LysM) domain family, were consecutively deleted, removed, or, alternatively, one additional repeat was added, without destroying the cell wall-hydrolyzing activity of the enzyme in vitro, although AcmA activity was reduced in all cases. In vivo, proteins containing no or only one repeat did not give rise to autolysis of lactococcal cells, whereas separation of the producer cells from the chains was incomplete. Exogenously added AcmA deletion derivatives carrying two repeats or four repeats bound to lactococcal cells, whereas the derivative with no or one repeat did not. In conclusion, these results show that AcmA needs three LysM domains for optimal peptidoglycan binding and biological functioning.

Acetylglucosaminidase↗

PubMed2005

Alternative lactose catabolic pathway in Lactococcus lactis IL1403.

In this study, we present a glimpse of the diversity of Lactococcus lactis subsp. lactis IL1403 beta-galactosidase phenotype-negative mutants isolated by negative selection on solid media containing cellobiose or lactose and X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside), and we identify several genes essential for lactose assimilation. Among these are ccpA (encoding catabolite control protein A), bglS (encoding phospho-beta-glucosidase), and several genes from the Leloir pathway gene cluster encoding proteins presumably essential for lactose metabolism. The functions of these genes were demonstrated by their disruption and testing of the growth of resultant mutants in lactose-containing media. By examining the ccpA and bglS mutants for phospho-beta-galactosidase activity, we showed that expression of bglS is not under strong control of CcpA. Moreover, this analysis revealed that although BglS is homologous to a putative phospho-beta-glucosidase, it also exhibits phospho-beta-galactosidase activity and is the major enzyme in L. lactis IL1403 involved in lactose hydrolysis.

Bacterial Proteins↗

PubMed2005

Autolysis of Lactococcus lactis is increased upon D-alanine depletion of peptidoglycan and lipoteichoic acids.

Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase (alr) mutant is strictly dependent on an external supply of D-Ala to be able to synthesize peptidoglycan and to incorporate D-Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when D-Ala is removed at mid-exponential growth. AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of D-Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the D-alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that D-alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking D-Ala has been reported in other bacterial species to have an improved capacity for autolysin binding. Mutation of dltD in L. lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, D-Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis. Second, reduced amounts of D-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.

Alanine↗

PubMed2005

Fructose utilization in Lactococcus lactis as a model for low-GC gram-positive bacteria: its regulator, signal, and DNA-binding site.

In addition to its role as carbon and energy source, fructose metabolism was reported to affect other cellular processes, such as biofilm formation by streptococci and bacterial pathogenicity in plants. Fructose genes encoding a 1-phosphofructokinase and a phosphotransferase system (PTS) fructose-specific enzyme IIABC component reside commonly in a gene cluster with a DeoR family regulator in various gram-positive bacteria. We present a comprehensive study of fructose metabolism in Lactococcus lactis, including a systematic study of fru mutants, global messenger analysis, and a molecular characterization of its regulation. The fru operon is regulated at the transcriptional level by both FruR and CcpA and at the metabolic level by inducer exclusion. The FruR effector is fructose-1-phosphate (F1P), as shown by combined analysis of transcription and measurements of the intracellular F1P pools in mutants either unable to produce this metabolite or accumulating it. The regulation of the fru operon by FruR requires four adjacent 10-bp direct repeats. The well-conserved organization of the fru promoter region in various low-GC gram-positive bacteria, including CRE boxes as well as the newly defined FruR motif, suggests that the regulation scheme defined in L. lactis could be applied to these bacteria. Transcriptome profiling of fruR and fruC mutants revealed that the effect of F1P and FruR regulation is limited to the fru operon in L. lactis. This result is enforced by the fact that no other targets for FruR were found in the available low-GC gram-positive bacteria genomes, suggesting that additional phenotypical effects due to fructose metabolism do not rely directly on FruR control, but rather on metabolism.

Amino Acid Sequence↗

PubMed2005

Probing direct interactions between CodY and the oppD promoter of Lactococcus lactis.

CodY of Lactococcus lactis MG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. These genes include pepN, pepC, opp-pepO1, and probably prtPM, pepX, and pepDA2, since the expression of the latter three genes relative to nitrogen availability is similar to that of the former. By means of in vitro DNA binding assays and DNase I footprinting techniques, we demonstrate that L. lactis CodY interacts directly with a region upstream of the promoter of its major target known so far, the opp system. Our results indicate that multiple molecules of CodY interact with this promoter and that the amount of bound CodY molecules is affected by the presence of branched-chain amino acids and not by GTP. Addition of these amino acids strongly affects the extent of the region protected by CodY in DNase I footprints. Random and site-directed mutagenesis of the upstream region of oppD yielded variants that were derepressed in a medium with an excess of nitrogen sources. Binding studies revealed the importance of specific bases in the promoter region required for recognition by CodY.

Amino Acids, Branched-Chain↗

PubMed2004

Two homologous oligopeptide binding protein genes (oppA) in Lactococcus lactis opp2 [corrected].

In previous studies, it has been shown that inactivation of opp or even oppA abolishes the capacity of Lactococcus lactis to utilize oligopeptides. We now show that the opp operon has been duplicated in L. lactis MG1363. The nucleotide sequence of the oppA and oppC homologues (appA and appC) and most of the oppB homologue (appB) indicate that the corresponding protein sequences are 83%, 92% and 91% identical, respectively. Inactivation of appA, via homologous recombination, as well as complementation studies were carried out to determine the possible function of appA in peptide utilization. As anticipated from studies with an oppA knock-out, peptide utilization was not impaired in an appA disruption mutant. Importantly, AppA expressed from a plasmid could restore the ability of oppA deletion mutants to utilize Leu-enkephalin, albeit with a lower efficiency than OppA. The differences in the ability to utilize this pentapeptide were not due to differences in expression levels but most likely reflect a different catalytic efficiency in oligopeptide utilization when AppA is used as ligand receptor.

Amino Acid Sequence↗

PubMed2004

Resistance of Gram-positive bacteria to nisin is not determined by lipid II levels.

Lipid II is essential for nisin-mediated pore formation at nano-molar concentrations. We tested whether nisin resistance could result from different Lipid II levels, by comparing the maximal Lipid II pool in Micrococcus flavus (sensitive) and Listeria monocytogenes (relatively insensitive) and their nisin-resistant variants, with a newly developed method. No correlation was observed between the maximal Lipid II pool and nisin sensitivity, as was further corroborated by using spheroplasts of nisin-resistant and wild-type strains of M. flavus, which were equally sensitive to nisin.

Anti-Bacterial Agents↗

PubMed2004

Genome2D: a visualization tool for the rapid analysis of bacterial transcriptome data.

Genome2D is a Windows-based software tool for visualization of bacterial transcriptome and customized datasets on linear chromosome maps constructed from annotated genome sequences. Genome2D facilitates the analysis of transcriptome data by using different color ranges to depict differences in gene-expression levels on a genome map. Such output format enables visual inspection of the transcriptome data, and will quickly reveal transcriptional units, without prior knowledge of expression level cutoff values. The compiled version of Genome2D is freely available for academic or non-profit use from http://molgen.biol.rug.nl/molgen/research/molgensoftware.php.

Bacterial Proteins↗

PubMed2004

Molecular Genetics Information System (MOLGENIS): alternatives in developing local experimental genomics databases.

MOTIVATION: Genomic research laboratories need adequate infrastructure to support management of their data production and research workflow. But what makes infrastructure adequate? A lack of appropriate criteria makes any decision on buying or developing a system difficult. Here, we report on the decision process for the case of a molecular genetics group establishing a microarray laboratory. RESULTS: Five typical requirements for experimental genomics database systems were identified: (i) evolution ability to keep up with the fast developing genomics field; (ii) a suitable data model to deal with local diversity; (iii) suitable storage of data files in the system; (iv) easy exchange with other software; and (v) low maintenance costs. The computer scientists and the researchers of the local microarray laboratory considered alternative solutions for these five requirements and chose the following options: (i) use of automatic code generation; (ii) a customized data model based on standards; (iii) storage of datasets as black boxes instead of decomposing them in database tables; (iv) loosely linking to other programs for improved flexibility; and (v) a low-maintenance web-based user interface. Our team evaluated existing microarray databases and then decided to build a new system, Molecular Genetics Information System (MOLGENIS), implemented using code generation in a period of three months. This case can provide valuable insights and lessons to both software developers and a user community embarking on large-scale genomic projects. AVAILABILITY: http://www.molgenis.nl

Computational Biology↗

PubMed2004

Subcellular sites for bacterial protein export.

Most bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA-YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the secretory protein pre-AmyQ, were analysed using green fluorescent protein fusions, immunostaining and/or immunogold labelling techniques. It is shown that SecA, SecY and (pre-)AmyQ are located at specific sites near and/or in the cytoplasmic membrane of Bacillus subtilis. The localization patterns of these proteins suggest that the Sec machinery is organized in spiral-like structures along the cell, with most of the translocases organized in specific clusters along these structures. However, this localization appears to be independent of the helicoidal structures formed by the actin-like cytoskeletal proteins, MreB or Mbl. Interestingly, the specific localization of SecA is dynamic, and depends on active translation. Moreover, reducing the phosphatidylglycerol phospholipids content in the bacterial membrane results in delocalization of SecA, suggesting the involvement of membrane phospholipids in the localization process. These data show for the first time that, in contrast to the recently reported uni-ExPortal site in the coccoïd Streptococcus pyogenes, multiple sites dedicated to protein export are present in the cytoplasmic membrane of rod-shaped B. subtilis.

Adenosine Triphosphatases↗

PubMed2004

ArgR and AhrC are both required for regulation of arginine metabolism in Lactococcus lactis.

The DNA binding proteins ArgR and AhrC are essential for regulation of arginine metabolism in Escherichia coli and Bacillus subtilis, respectively. A unique property of these regulators is that they form hexameric protein complexes, mediating repression of arginine biosynthetic pathways as well as activation of arginine catabolic pathways. The gltS-argE operon of Lactococcus lactis encodes a putative glutamate or arginine transport protein and acetylornithine deacetylase, which catalyzes an important step in the arginine biosynthesis pathway. By random integration knockout screening we found that derepression mutants had ISS1 integrations in, among others, argR and ahrC. Single as well as double regulator deletion mutants were constructed from Lactococcus lactis subsp. cremoris MG1363. The three arginine biosynthetic operons argCJDBF, argGH, and gltS-argE were shown to be repressed by the products of argR and ahrC. Furthermore, the arginine catabolic arcABD1C1C2TD2 operon was activated by the product of ahrC but not by that of argR. Expression from the promoter of the argCJDBF operon reached similar levels in the single mutants and in the double mutant, suggesting that the regulators are interdependent and not able to complement each other. At the same time they also appear to have different functions, as only AhrC is involved in activation of arginine catabolism. This is the first study where two homologous arginine regulators are shown to be involved in arginine regulation in a prokaryote, representing an unusual mechanism of regulation.

Amino Acid Sequence↗