Biomedical subjects

Chris D den Hengst

Publications and source records attributed to Chris D den Hengst.

6 recordsLinked to original sources

PubMed2006

Expression of transcription activator ComK of Bacillus subtilis in the heterologous host Lactococcus lactis leads to a genome-wide repression pattern: a case study of horizontal gene transfer.

Horizontal gene transfer (HGT) is generally considered a possible mechanism by which bacteria acquire new genetic properties. Especially when pathogenicity genes are involved, HGT might have important consequences for humans. In this report we describe a case study of HGT in which a transcriptional activator, ComK of Bacillus subtilis, was introduced into a heterologous host species, Lactococcus lactis. ComK is the central regulator of competence development, activating transcription by binding to a ComK-binding site, a so-called K-box. Interestingly, L. lactis does not contain a comK gene, but it does contain almost 400 putatively functional K-boxes, as well as homologues of a number of competence genes. In this study, the effect of HGT of B. subtilis comK into L. lactis was investigated by determining the effects on the transcription profile using DNA microarray analyses. Production of wild-type ComK was shown to stimulate the transcription of 89 genes and decrease the expression of 114 genes. Notably, potential direct effects (i.e., genes preceded by a K-box) were found mainly among repressed genes, suggesting that ComK functions as a repressor in L. lactis. This is a remarkable difference between L. lactis and B. subtilis, in which ComK almost exclusively activates transcription. Additional DNA microarray analyses with a transcription activation-deficient but DNA-binding ComK variant, ComKDeltaC25, demonstrated that there were similar effects on gene regulation with this variant and with wild-type ComK, confirming that the direct effects of ComK result from interference with normal transcription through binding to available K-boxes. This study demonstrates that horizontal gene transfer can have dramatic effects that are very different than those that are expected on basis of the original functionality of a gene.

Bacillus subtilis↗

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↗

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

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↗

PubMed2002

Transcriptome analysis and related databases of Lactococcus lactis.

Several complete genome sequences of Lactococcus lactis and their annotations will become available in the near future, next to the already published genome sequence of L. lactis ssp. lactis IL 1403. This will allow intraspecies comparative genomics studies as well as functional genomics studies aimed at a better understanding of physiological processes and regulatory networks operating in lactococci. This paper describes the initial set-up of a DNA-microarray facility in our group, to enable transcriptome analysis of various Gram-positive bacteria, including a ssp. lactis and a ssp. cremoris strain of Lactococcus lactis. Moreover a global description will be given of the hardware and software requirements for such a set-up, highlighting the crucial integration of relevant bioinformatics tools and methods. This includes the development of MolGenIS, an information system for transcriptome data storage and retrieval, and LactococCye, a metabolic pathway/genome database of Lactococcus lactis.

Databases, Nucleic Acid↗