Papers by Wilfried Meijer
Acta Crystallographica Section D: Structural Biology, Mar 1, 2023
Transcriptional regulation usually requires the action of several proteins that either repress or... more Transcriptional regulation usually requires the action of several proteins that either repress or activate a promotor of an open reading frame. These proteins can counteract each other, thus allowing tight regulation of the transcription of the corresponding genes, where tight repression is often linked to DNA looping or cross-linking. Here, the tetramerization domain of the bacterial gene repressor Rco from Bacillus subtilis plasmid pLS20 (Rco pLS20) has been identified and its structure is shown to share high similarity to the tetramerization domain of the well known p53 family of human tumor suppressors, despite lacking clear sequence homology. In Rco pLS20 , this tetramerization domain is responsible for inducing DNA looping, a process that involves multiple tetramers. In accordance, it is shown that Rco pLS20 can form octamers. This domain was named TetD loop and its occurrence was identified in other Bacillus species. The TetD loop fold was also found in the structure of a transcriptional repressor from Salmonella phage SPC32H. It is proposed that the TetD loop fold has evolved through divergent evolution and that the TetD loop originates from a common ancestor predating the occurrence of multicellular life.
Bacillus subtilis contains at least three chromosomally-encoded type I signal peptidases (SPases;... more Bacillus subtilis contains at least three chromosomally-encoded type I signal peptidases (SPases; SipS, SipT, and SipU), which remove signal peptides from secretory proteins. In addition, certain B. subtilis (natto) strains contain plasmid-encoded type I SPases (SipP). The known type I SPases from B. subtilis show a high degree of similarity to SPases from related bacilli and Staphylococcus aureus and, to a lesser extent, to SPases from other organisms. In addition, the putative active site region of the Bacillus SPases shows similarity to the corresponding region of LexA-like proteases, suggesting that the type I SPases employ a serine-lysine catalytic dyed. Unlike the type I SPase of Escherichia coli, Sips, SipT and SipU are neither essential for protein secretion, nor for viability of the cell. Although non-essential, Sips is an important factor for efficient protein secretion. SipS is transcribed in a growth phase-and medium-dependent manner. Under some conditions, transcription of sipS is controlled by the DegS-DegU two-component regulatory system, indicating that expression of sipS is determined by the same factors that control the expression of most genes for secreted degradative enzymes. These observations suggest thats. subtilis can modulate its capacity and specificity for protein secretion through controlled expression of sipS.
Molecular Microbiology, Mar 1, 1996
ABSTRACT
Research Square (Research Square), May 2, 2023
Journal of Biological Chemistry, Nov 1, 2004
The Bacillus subtilis phage 29-encoded membrane protein p16.7 is one of the few proteins known to... more The Bacillus subtilis phage 29-encoded membrane protein p16.7 is one of the few proteins known to be involved in prokaryotic membrane-associated DNA replication. Protein p16.7 contains an N-terminal transmembrane domain responsible for membrane localization. A soluble variant lacking the N-terminal membrane anchor, p16.7A, forms dimers in solution, binds to DNA, and has affinity for the 29 terminal protein. Here we show that the soluble N-terminal half of p16.7A can form a dimeric coiled coil. However, a second domain, located in the C-terminal half of the protein, has been characterized as being the main domain responsible for p16.7 dimerization. This 70-residue C-terminal domain, named p16.7C, also constitutes the functional part of the protein as it binds to DNA and terminal protein. Sequence alignments, secondary structure predictions, and spectroscopic analyses suggest that p16.7C is evolutionarily related to DNA binding homeodomains, present in many eukaryotic transcriptional regulator proteins. Based on the results, a structural model of p16.7 is presented.
Current Biology, Nov 1, 2021
Some Bacillus-infecting bacteriophages use a peptide-based communication system, termed arbitrium... more Some Bacillus-infecting bacteriophages use a peptide-based communication system, termed arbitrium, to coordinate the lysis-lysogeny decision. In this system, the phage produces AimP peptide during the lytic cycle. Once internalized by the host cell, AimP binds to the transcription factor AimR, reducing aimX expression and promoting lysogeny. Although these systems are present in a variety of mobile genetic elements, their role in the phage life cycle has only been characterized in phage phi3T during phage infection. Here, using the B. subtilis SPβ prophage, we show that the arbitrium system is also required for normal prophage induction. Deletion of the aimP gene increased phage reproduction, although the aimR deletion significantly reduced the number of phage particles produced after prophage induction. Moreover, our results indicated that AimR is involved in a complex network of regulation and brought forward two new players in the SPβ lysis-lysogeny decision system, YopN and the phage repressor YopR. Importantly, these proteins are encoded in an operon, the function of which is conserved across all SPβ-like phages encoding the arbitrium system. Finally, we obtained mutant phages in the arbitrium system, which behaved almost identically to the wild-type (WT) phage, indicating that the arbitrium system is not essential in the laboratory but is likely beneficial for phage fitness in nature. In support of this, by possessing a functional arbitrium system, the SPβ phage can optimize production of infective particles while also preserving the number of cells that survive after prophage induction, a strategy that increases phage persistence in nature.
Environmental Microbiology, Jul 11, 2012
SummaryUnder certain growth conditions, Bacillus subtilis can develop natural competence, the sta... more SummaryUnder certain growth conditions, Bacillus subtilis can develop natural competence, the state in which it is able to bind, adsorb and incorporate exogenous DNA. Development of competence is a bistable process and is subject to complex regulation. Rok is a repressor of the key transcriptional activator of competence genes, comK, and limits the size of the subpopulation that develops competence. Here we report the finding that the large conjugative B. subtilis plasmid pLS20 harbours a rok homologue rokLS20. Although the deduced product of rokLS20 is considerably shorter than the chromosomally encoded Rok protein, we show that ectopic expression of the plasmid‐encoded RokLS20 leads to inhibition of competence by repressing comK, and that the effects of the plasmid and chromosomally encoded Rok proteins are additive. We also show that pLS20 inhibits competence in a rokLS20‐dependent manner and that purified RokLS20 preferentially binds to the comK promoter. By analysing the available databases we identified several additional rok‐like genes. These putative rok genes can be divided into two groups and we propose that rokLS20 is the prototype of a newly identified subgroup of nine rok genes. Finally, we discuss the possible role of the plasmid‐located rok and its relatedness with other rok genes.
Research in Microbiology, 1991
Nucleic Acids Research
Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complex... more Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complexes associated with an antitermination factor can bypass multiple transcription termination signals regardless of their sequences. However, to avoid compromising transcriptional regulation of downstream regions, the terminator at the end of the operon needs to be resistant to antitermination. So far, no studies on the mechanism of resistance to antitermination have been reported. The recently discovered conAn P-AT system is composed of two components that are encoded at the start of many conjugation operons on plasmids of Gram-positive bacteria. Here we report the identification of a conAn-resistant terminator, named TerR, in the conjugation operon of the Bacillus subtilis plasmid pLS20, re-defining the end of the conjugation operon. We investigated the various characteristics of TerR and show that its extraordinary long stem is the determining feature for resistance to antitermination. T...
BIO-PROTOCOL
Geobacillus kaustophilus, a thermophilic Gram-positive bacterium, is an attractive host for the d... more Geobacillus kaustophilus, a thermophilic Gram-positive bacterium, is an attractive host for the development of high-temperature bioprocesses. However, its reluctance against genetic manipulation by standard methodologies hampers its exploitation. Here, we describe a simple methodology in which an artificial DNA segment on the chromosome of Bacillus subtilis can be transferred via pLS20-mediated conjugation resulting in subsequent integration in the genome of G. kaustophilus. Therefore, we have developed a transformation strategy to design an artificial DNA segment on the chromosome of B. subtilis and introduce it into G. kaustophilus. The artificial DNA segment can be freely designed by taking advantage of the plasticity of the B. subtilis genome and combined with the simplicity of pLS20 conjugation transfer. This transformation strategy would adapt to various Gram-positive bacteria other than G. kaustophilus.
Differential Spo0A-mediated effects on transcription and replication of the related Bacillus subt... more Differential Spo0A-mediated effects on transcription and replication of the related Bacillus subtilis phages Nf and r29 explain their different behaviours in vivo
Academic Thesis, State University of Groningen …, 1995
Nucleic Acids Research
Nucleoid-associated proteins (NAPs) play a central role in chromosome organization and environmen... more Nucleoid-associated proteins (NAPs) play a central role in chromosome organization and environment-responsive transcription regulation. The Bacillus subtilis-encoded NAP Rok binds preferentially AT-rich regions of the genome, which often contain genes of foreign origin that are silenced by Rok binding. Additionally, Rok plays a role in chromosome architecture by binding in genomic clusters and promoting chromosomal loop formation. Based on this, Rok was proposed to be a functional homolog of E. coli H-NS. However, it is largely unclear how Rok binds DNA, how it represses transcription and whether Rok mediates environment-responsive gene regulation. Here, we investigated Rok's DNA binding properties and the effects of physico-chemical conditions thereon. We demonstrate that Rok is a DNA bridging protein similar to prototypical H-NS-like proteins. However, unlike these proteins, the DNA bridging ability of Rok is not affected by changes in physico-chemical conditions. The DNA bind...
<p><b>A</b>. Schematic representation of the region corresponding to Fragment V... more <p><b>A</b>. Schematic representation of the region corresponding to Fragment V, which encompasses promoters <i>P<sub>r</sub></i>/<i>P<sub>c</sub></i> and the identified repeated motif that form (part of) the binding site for Rco<sub>LS20</sub>. Top line. Position of promoters <i>P<sub>c</sub></i> and <i>P<sub>r</sub></i> are indicated in blue and red, respectively. The position of the unique <i>Eco</i>RI site is indicated. A 13 bp long direct repeat (5′-TCAGTGAAAAAAA-3′) is indicated with rightward directed red arrows. The leftward-directed arrow indicates the position of the complementary 9 bp sequence 5′-TTTCACTGA-3′. Second line (Fragment V). Arrows indicate the positions of the identified motifs a1–a7 and b1–b4. Motifs present on the upper and the lower strand are shown in green and purple, respectively. Third and fourth line show identified motifs present on Fragment III_A and XII_A, respectively. Black, dark grey, grey and light grey indicate motifs identical to the consensus sequence or deviating at one, two or three positions, respectively. <b>B</b>. An alignment of the nucleotide sequences of the eleven identified motifs and their flanking sequences. Names according to the nomenclature in “A” are given together with information on the strand and the region. Sequences corresponding to the consensus sequence of the motif are given in bold. <b>C</b>. A representation of the consensus motif generated by Weblogo <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004733#pgen.1004733-Crooks1" target="_blank">[73]</a>. The size of each nucleotide corresponds to the frequency with which that nucleotide is observed in that position.</p
<p><b>A</b>. Determination of promoter <i>P<sub>c</sub></i... more <p><b>A</b>. Determination of promoter <i>P<sub>c</sub></i> and <i>P<sub>r</sub></i> sequences by deletion analysis and primer extension. pLS20cat containing cells harvested at the end of the exponential growth phase were processed to isolate their total RNA, which was used in primer extension assays as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004733#s4" target="_blank">Materials and Methods</a>. Features of the promoter <i>P<sub>c</sub></i> are shown above the sequence. The dotted vertical lines and black straight lines indicate the 5′ end points of the transcriptional <i>lacZ</i> fusions present in strains GR68 and GR70, displaying and not displaying promoter activity, respectively. The core promoter and the putative upstream UP element is indicated by a light blue box; the −35 and −10 hexamers, and the extended −10 motif are indicated with dark blue and green boxes, respectively. The transcription start site determined by primer extension and the direction of transcription are indicated with the corresponding encircled base and a black bent arrow. The thin grey bent arrow corresponds to the 3′ end point of the smaller extension product that coincides with the start of an inverted repeat which is marked with a pair of thin blue arrows above the sequence. Features of promoter <i>P<sub>r</sub></i> are shown below the sequence. The dotted vertical lines and the black straight lines indicate the 3′ end points of the transcriptional fusions with <i>lacZ</i> reporter present in strains GR82 and GR116, displaying and not displaying promoter activity, respectively. The deduced position of the <i>P<sub>r</sub></i> core promoter, and the −35 and −10 boxes are indicated with orange and red boxes, respectively (see text). The transcription start site determined by primer extension and the direction of transcription are indicated with the corresponding encircled base and a black bent arrow. <b>B</b>. Primer extension to determine the transcription start sites of promoters <i>P<sub>c</sub></i> (left panel) and <i>P<sub>r</sub></i> (right panel). The cDNA products of the primer extension reactions are indicated with bent arrows (lane P). Free lane in which no sample was run is indicated with “−”. Lanes M, M1 and M2 correspond to [G+A] chemical sequencing reactions of a short 230 bp DNA fragment corresponding to the studied pLS20cat region obtained by PCR amplification as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004733#s4" target="_blank">Materials and Methods</a>. In the case of the <i>P<sub>c</sub></i> promoter, a smaller extension product with a relatively strong signal was observed 37 bp downstream of the extension product shown. The longer extension product most likely reflects the correct transcription start site based on the following arguments. First, it is known that AMV reverse transcriptase prematurely terminates cDNA synthesis when reaching a stem loop in the RNA, and that the prematurely terminated molecules map at the bottom of the secondary structure <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004733#pgen.1004733-Shimomaye1" target="_blank">[71]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004733#pgen.1004733-Loreau1" target="_blank">[72]</a>. The position of the strong signal coincides with the 3′ end of an inverted repeat (indicated in Fig. 3A). Second, no putative core promoter sequences are evident upstream of the 5′ position of the shorter extension product. Third, if the stronger signal corresponds to the transcription start site, the responsible promoter would be present on Fragment VIII<sub>c</sub> used for the transcriptional <i>lacZ</i> fusion in strain GR70. However, no promoter activity was observed with this strain (see text). And fourth, the transcription start site based on the longer extension product corroborates the RNAseq data. <b>C</b>. Schematic overview of RNAseq expression data of pLS20cat genes <i>rco<sub>LS20</sub></i> and <i>28</i> under conditions with (top panel) and without overexpression of <i>rco<sub>LS20</sub></i> (lower panel). The amount of right and leftward “reads”, given in green and red, respectively, are presented on a log2 scale. The positions of the divergently oriented genes <i>rco<sub>LS20</sub></i> and <i>28</i> are indicated on the top with a red and green arrow, respectively. Dotted lines and black arrows indicate the approximate start sites of the divergent transcripts driven by the <i>P<sub>c</sub></i> and <i>P<sub>r</sub></i> promoters.</p
Uploads
Papers by Wilfried Meijer