Kluyver Center for Genomics of Industrial Fermentation

Delft, Netherlands

Kluyver Center for Genomics of Industrial Fermentation

Delft, Netherlands
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de Jong I.G.,University of Groningen | Veening J.-W.,University of Groningen | Kuipers O.P.,Kluyver Center for Genomics of Industrial Fermentation
Environmental Microbiology | Year: 2012

How cells dynamically respond to fluctuating environmental conditions depends on the architecture and noise of the underlying genetic circuits. Most work characterizing stress pathways in the model bacterium Bacillus subtilis has been performed on bulk cultures using ensemble assays. However, investigating the single cell response to stress is important since noise might generate significant phenotypic heterogeneity. Here, we study the stress response to carbon source starvation and compare both population and single cell data. Using a top-down approach, we investigate the transcriptional dynamics of various stress-related genes of B. subtilis in response to carbon source starvation and to increased cell density. Our data reveal that most of the tested gene-regulatory networks respond highly heterogeneously to starvation and cells show a large degree of variation in gene expression. The level of highly dynamic diversification within B. subtilis populations under changing environments reflects the necessity to study cells at the single cell level. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.


Boonstra M.,University of Groningen | de Jong I.G.,University of Groningen | Scholefield G.,Northumbria University | Murray H.,Northumbria University | And 3 more authors.
Molecular Microbiology | Year: 2013

When starved, Bacillus subtilis cells can enter the developmental programme of endospore formation by activation of the master transcriptional regulator Spo0A. Correct chromosome copy number is crucial for the production of mature and fully resistant spores. The production and maintenance of one chromosome for the mother cell and one copy for the forespore requires accurate co-ordination between DNA replication and initiation of sporulation. Here, we show that Spo0A regulates chromosome copy number by directly binding to a number of Spo0A binding sites that are present near the origin of replication (oriC). We demonstrate that cells lacking three specific Spo0A binding sites at oriC display increased chromosome copy numbers when sporulation is induced. Our data support the hypothesis that Spo0A directly controls DNA replication during sporulation by binding to oriC. © 2013 Blackwell Publishing Ltd.


Khusainov R.,University of Groningen | Kuipers O.P.,University of Groningen | Kuipers O.P.,Kluyver Center for Genomics of Industrial Fermentation
PLoS ONE | Year: 2013

Precursor nisin is a model posttranslationally modified precursor lantibiotic that can be structurally divided into a leader peptide sequence and a modifiable core peptide part. The nisin core peptide clearly plays an important role in the precursor nisin - nisin modification enzymes interactions, since it has previously been shown that the construct containing only the nisin leader sequence is not sufficient to pull-down the nisin modification enzymes NisB and NisC. Serines and threonines in the core peptide part are the residues that NisB specifically dehydrates, and cysteines are the residues that NisC stereospecifically couples to the dehydrated amino acids. Here, we demonstrate that increasing the number of negatively charged residues in the core peptide part of precursor nisin, which are absent in wild-type nisin, does not abolish binding of precursor nisin to the modification enzymes NisB and NisC, but dramatically decreases the antimicrobial potency of these nisin mutants. An unnatural precursor nisin variant lacking all serines and threonines in the core peptide part and an unnatural precursor nisin variant lacking all cysteines in the core peptide part still bind the nisin modification enzymes NisB and NisC, suggesting that these residues are not essential for direct interactions with the nisin modification enzymes NisB and NisC. These results are important for lantibiotic engineering studies. © 2013 Khusainov and Kuipers.


Kovacs A.T.,University of Groningen | Kuipers O.P.,University of Groningen | Kuipers O.P.,Kluyver Center for Genomics of Industrial Fermentation
Journal of Bacteriology | Year: 2011

Transcriptome analysis of a Bacillus subtilis rok strain that showed reduced complex colony structure formation revealed significant downregulation of the yuaB gene. Overexpression of yuaB restored structure formation in the rok strain. We show that transcription of yuaB is indirectly regulated by Rok, independently from its previously described AbrB-dependent regulation. Copyright © 2011, American Society for Microbiology. All Rights Reserved.


De Jong I.G.,University of Groningen | Haccou P.,Leiden University | Kuipers O.P.,University of Groningen | Kuipers O.P.,Kluyver Center for Genomics of Industrial Fermentation
BioEssays | Year: 2011

Bacteria have developed an impressive ability to survive and propagate in highly diverse and changing environments by evolving phenotypic heterogeneity. Phenotypic heterogeneity ensures that a subpopulation is well prepared for environmental changes. The expression bet hedging is commonly (but often incorrectly) used by molecular biologists to describe any observed phenotypic heterogeneity. In evolutionary biology, however, bet hedging denotes a risk-spreading strategy displayed by isogenic populations that evolved in unpredictably changing environments. Opposed to other survival strategies, bet hedging evolves because the selection environment changes and favours different phenotypes at different times. Consequently, in bet hedging populations all phenotypes perform differently well at any time, depending on the selection pressures present. Moreover, bet hedging is the only strategy in which temporal variance of offspring numbers per individual is minimized. Our paper aims to provide a guide for the correct use of the term bet hedging in molecular biology. Bet hedging describes a risk-spreading strategy displayed by isogenic populations that evolved in unpredictably changing environments and is the only strategy in which temporal variance of offspring numbers per individual is minimized. © 2011 WILEY Periodicals, Inc.


Khusainov R.,University of Groningen | Heils R.,University of Groningen | Lubelski J.,University of Groningen | Moll G.N.,LanthioPep | And 2 more authors.
Molecular Microbiology | Year: 2011

Although nisin is a model lantibiotic, our knowledge of the specific interactions of prenisin with its modification enzymes remains fragmentary. Here, we demonstrate that the nisin modification enzymes NisB and NisC can be pulled down in vitro from Lactococcus lactis by an engineered His-tagged prenisin. This approach enables us to determine important intermolecular interactions of prenisin with its modification machinery within L.lactis. We demonstrate that (i) NisB has stronger interactions with precursor nisin than NisC has, (ii) deletion of the propeptide part keeping the nisin leader intact leads to a lack of binding, (iii) NisB point mutants of highly conserved residues W616, F342A, Y346F and P639A are still able to dehydrate prenisin, (iv) NisB Δ(77-79)Y80F mutant decreased the levels of NisB-prenisin interactions and resulted in unmodified prenisin, (v) substitution of an active site residue H331A in NisC leads to higher amounts of the co-purified complex, (vi) NisB is present in the form of a dimer, and (vii) the region FNLD (-18 to -15) of the leader is an important site for binding not only to NisB, but also to NisC. © 2011 Blackwell Publishing Ltd.


de Jong A.,University of Groningen | van Heel A.J.,University of Groningen | Kok J.,University of Groningen | Kuipers O.P.,University of Groningen | Kuipers O.P.,Kluyver Center for Genomics of Industrial Fermentation
Nucleic Acids Research | Year: 2010

Mining bacterial genomes for bacteriocins is a challenging task due to the substantial structure and sequence diversity, and generally small sizes, of these antimicrobial peptides. Major progress in the research of antimicrobial peptides and the ever-increasing quantities of genomic data, varying from (un)finished genomes to meta-genomic data, led us to develop the significantly improved genome mining software BAGEL2, as a follow-up of our previous BAGEL software. BAGEL2 identifies putative bacteriocins on the basis of conserved domains, physical properties and the presence of biosynthesis, transport and immunity genes in their genomic context. The software supports parameter-free, class-specific mining and has high-throughput capabilities. Besides building an expert validated bacteriocin database, we describe the development of novel Hidden Markov Models (HMMs) and the interpretation of combinations of HMMs via simple decision rules for prediction of bacteriocin (sub-)classes. Furthermore, the genetic context is automatically annotated based on (combinations of) PFAM domains and databases of known context genes. The scoring system was fine-tuned using expert knowledge on data derived from screening all bacterial genomes currently available at the NCBI. BAGEL2 is freely accessible at http://bagel2.molgenrug.nl. © The Author(s) 2010. Published by Oxford University Press.


Kovalchuk A.,Zernike Institute for Advanced Materials | Kovalchuk A.,Kluyver Center for Genomics of Industrial Fermentation | Driessen A.J.M.,Zernike Institute for Advanced Materials | Driessen A.J.M.,Kluyver Center for Genomics of Industrial Fermentation
BMC Genomics | Year: 2010

Background: The superfamily of ABC proteins is among the largest known in nature. Its members are mainly, but not exclusively, involved in the transport of a broad range of substrates across biological membranes. Many contribute to multidrug resistance in microbial pathogens and cancer cells. The diversity of ABC proteins in fungi is comparable with those in multicellular animals, but so far fungal ABC proteins have barely been studied.Results: We performed a phylogenetic analysis of the ABC proteins extracted from the genomes of 27 fungal species from 18 orders representing 5 fungal phyla thereby covering the most important groups. Our analysis demonstrated that some of the subfamilies of ABC proteins remained highly conserved in fungi, while others have undergone a remarkable group-specific diversification. Members of the various fungal phyla also differed significantly in the number of ABC proteins found in their genomes, which is especially reduced in the yeast S. cerevisiae and S. pombe.Conclusions: Data obtained during our analysis should contribute to a better understanding of the diversity of the fungal ABC proteins and provide important clues about their possible biological functions. © 2010 Kovalchuk and Driessen; licensee BioMed Central Ltd.


Siezen R.J.,Kluyver Center for Genomics of Industrial Fermentation | Siezen R.J.,Radboud University Nijmegen | Siezen R.J.,Netherlands Bioinformatics Center | van Hylckama Vlieg J.E.T.,Kluyver Center for Genomics of Industrial Fermentation | van Hylckama Vlieg J.E.T.,Danone Inc.
Microbial Cell Factories | Year: 2011

In the past decade it has become clear that the lactic acid bacterium Lactobacillus plantarum occupies a diverse range of environmental niches and has an enormous diversity in phenotypic properties, metabolic capacity and industrial applications. In this review, we describe how genome sequencing, comparative genome hybridization and comparative genomics has provided insight into the underlying genomic diversity and versatility of L. plantarum. One of the main features appears to be genomic life-style islands consisting of numerous functional gene cassettes, in particular for carbohydrates utilization, which can be acquired, shuffled, substituted or deleted in response to niche requirements. In this sense, L. plantarum can be considered a "natural metabolic engineer". © 2011 Siezen and van Hylckama Vlieg; licensee BioMed Central Ltd.


Bachmann H.,NIZO Food Research | Bachmann H.,Kluyver Center for Genomics of Industrial Fermentation | Bachmann H.,VU University Amsterdam | Starrenburg M.J.C.,NIZO Food Research | And 10 more authors.
Genome Research | Year: 2012

Experimental evolution is a powerful approach to unravel how selective forces shape microbial genotypes and phenotypes. To this date, the available examples focus on the adaptation to conditions specific to the laboratory. The lactic acid bacterium Lactococcus lactis naturally occurs on plants and in dairy environments, and it is proposed that dairy strains originate from the plant niche. Here we investigate the adaptation of a L. lactis strain isolated from a plant to a dairy niche by propagating it for 1000 generations in milk. Two out of three independently evolved strains displayed significantly increased acidification rates and biomass yields in milk. Genome resequencing, revealed six, seven, and 28 mutations in the three strains, including point mutations in loci related to amino acid biosynthesis and transport and in the gene encoding MutL, which is involved in DNA mismatch repair. Two strains lost a conjugative transposon containing genes important in the plant niche but dispensable in milk. A plasmid carrying an extracellular protease was introduced by transformation. Although improving growth rate and growth yield significantly, the plasmid was rapidly lost. Comparative transcriptome and phenotypic analyses confirmed that major physiological changes associated with improved growth in milk relate to nitrogen metabolism and the loss or down-regulation of several pathways involved in the utilization of complex plant polymers. Reproducing the transition from the plant to the dairy niche through experimental evolution revealed several genome, transcriptome, and phenotype signatures that resemble those seen in strains isolated from either niche. © 2012 by Cold Spring Harbor Laboratory Press.

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