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23 Publications visible to you, out of a total of 23
What makes the lac-pathway switch: identifying the fluctuations that trigger phenotype switching in gene regulatory systems

Abstract (Expand)

Multistable gene regulatory systems sustain different levels of gene expression under identical external conditions. Such multistability is used to encode phenotypic states in processes including … nutrient uptake and persistence in bacteria, fate selection in viral infection, cell-cycle control and development. Stochastic switching between different phenotypes can occur as the result of random fluctuations in molecular copy numbers of mRNA and proteins arising in transcription, translation, transport and binding. However, which component of a pathway triggers such a transition is generally not known. By linking single-cell experiments on the lactose-uptake pathway in E. coli to molecular simulations, we devise a general method to pinpoint the particular fluctuation driving phenotype switching and apply this method to the transition between the uninduced and induced states of the lac-genes. We find that the transition to the induced state is not caused only by the single event of lac-repressor unbinding, but depends crucially on the time period over which the repressor remains unbound from the lac-operon. We confirm this notion in strains with a high expression level of the lac-repressor (leading to shorter periods over which the lac-operon remains unbound), which show a reduced switching rate. Our techniques apply to multistable gene regulatory systems in general and allow to identify the molecular mechanisms behind stochastic transitions in gene regulatory circuits.

Editor:

Date Published: 24th Sep 2014

Publication Type: Not specified

Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae

Abstract (Expand)

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such … pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in 'transcription traffic jams' on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.

Authors: , , M. Herber, L. Attaiech, , , S. Klumpp, ,

Date Published: 6th Sep 2014

Publication Type: Not specified

Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging

Abstract (Expand)

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell … biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two "superfolder" GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

Authors: W. Overkamp, K. Beilharz, R. Detert Oude Weme, A. Solopova, H. Karsens, A. Kovacs, J. Kok, ,

Date Published: 16th Aug 2013

Publication Type: Not specified

GraphAlignment: Bayesian pairwise alignment of biological networks

Abstract (Expand)

ABSTRACT: BACKGROUND: With increased experimental availability and accuracy of bio-molecular networks, tools for their comparative and evolutionary analysis are needed. A key component for such studies … is the alignment of networks. RESULTS: We introduce the Bioconductor package GraphAlignment for pairwise alignment of bio-molecular networks. The alignment incorporates information both from network vertices and network edges and is based on an explicit evolutionary model, allowing inference of all scoring parameters directly from empirical data. We compare the performance of our algorithm to an alternative algorithm, Graemlin 2.0.On simulated data, GraphAlignment outperforms Graemlin 2.0 in several benchmarks except for computational complexity. When there is little or no noise in the data, GraphAlignment is slower than Graemlin 2.0. It is faster than Graemlin 2.0 when processing noisy data containing spurious vertex associations. Its typical case complexity grows approximately as O(N^2.6). On empirical bacterial protein-protein interaction networks (PIN) and gene co-expression networks, GraphAlignment outperforms Graemlin 2.0 with respect to coverage and specificity, albeit by a small margin. On large eukaryotic PIN, Graemlin 2.0 outperforms GraphAlignment. CONCLUSIONS: The GraphAlignment algorithm is robust to spurious vertex associations, correctly resolves paralogs, and shows very good performance in identification of homologous vertices defined by high vertex and/or interaction similarity.

Authors: Michal Kolar, Jörn Meier, Ville Mustonen, Michael Lässig,

Date Published: 21st Nov 2012

Publication Type: Not specified

Single cell analysis of gene expression patterns during carbon starvation in Bacillus subtilis reveals large phenotypic variation

Abstract (Expand)

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.

Editor:

Date Published: 4th Oct 2012

Publication Type: Not specified

Multiple active centers of multi-subunit RNA polymerases

Abstract (Expand)

The active center of multi-subunit RNA polymerase consists of two modules, the Mg(2+) module, holding the catalytic Mg(2+) ion, and a module made of a flexible domain, the Trigger Loop. Uniquely, the … TL module can be substituted by alternative modules, thus changing the catalytic properties of the active center.

Authors: , Mohammad Roghanian,

Date Published: 10th Jul 2012

Publication Type: Not specified

Hypothesis: emergence of translation as a result of RNA helicase evolution

Abstract (Expand)

The origin of translation and the genetic code is one of the major mysteries of evolution. The advantage of templated protein synthesis could have been achieved only when the translation apparatus had … already become very complex. This means that the translation machinery, as we know it today, must have evolved towards some different essential function that subsequently sub-functionalised into templated protein synthesis. The hypothesis presented here proposes that translation originated as the result of evolution of a primordial RNA helicase, which has been essential for preventing dying out of the RNA organism in sterile double-stranded form. This hypothesis emerges because modern ribosome possesses RNA helicase activity that likely dates back to the RNA world. I hypothesise that codon-anticodon interactions of tRNAs with mRNA evolved as a mechanism used by RNA helicase, the predecessor of ribosomes, to melt RNA duplexes. In this scenario, peptide bond formation emerged to drive unidirectional movement of the helicase via a molecular ratchet mechanism powered by Brownian motion. I propose that protein synthesis appeared as a side product of helicase activity. The first templates for protein synthesis were functional RNAs (ribozymes) that were unwound by the helicase, and the first synthesised proteins were of random or non-sense sequence. I further suggest that genetic code emerged to avoid this randomness. The initial genetic code thus emerged as an assignment of amino acids to codons according to the sequences of the pre-existing RNAs to take advantage of the side products of RNA helicase function.

Editor:

Date Published: 28th Apr 2012

Publication Type: Not specified

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

Abstract (Expand)

How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type … Ser/Thr kinase of S. pneumoniae, StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.

Authors: Katrin Beilharz, Linda Nováková, Daniela Fadda, Pavel Branny, Orietta Massidda,

Date Published: 21st Mar 2012

Publication Type: Not specified

In vitro experimental system for analysis of transcription-translation coupling

Abstract (Expand)

Transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. During transcription-translation, both machineries mutually affect each others' … functions, which is important for regulation of gene expression. Analysis of interactions between RNA polymerase (RNAP) and the ribosome, however, are limited due to the lack of an in vitro experimental system. Here, we report the development of an in vitro transcription coupled to translation system assembled from purified components. The system allows controlled stepwise transcription and simultaneous stepwise translation of the nascent RNA, and permits investigation of the interactions of RNAP with the ribosome, as well as the effects of translation on transcription and transcription on translation. As an example of usage of this experimental system, we uncover complex effects of transcription-translation coupling on pausing of transcription.

Authors: Daniel Castro-Roa,

Date Published: 3rd Jan 2012

Publication Type: Not specified

Factor-independent transcription pausing caused by recognition of the RNA-DNA hybrid sequence

Abstract (Expand)

Pausing of transcription is an important step of regulation of gene expression in bacteria and eukaryotes. Here we uncover a factor-independent mechanism of transcription pausing, which is determined … by the ability of the elongating RNA polymerase to recognize the sequence of the RNA-DNA hybrid. We show that, independently of thermodynamic stability of the elongation complex, RNA polymerase directly 'senses' the shape and/or identity of base pairs of the RNA-DNA hybrid. Recognition of the RNA-DNA hybrid sequence delays translocation by RNA polymerase, and thus slows down the nucleotide addition cycle through 'in pathway' mechanism. We show that this phenomenon is conserved among bacterial and eukaryotic RNA polymerases, and is involved in regulatory pauses, such as a pause regulating the production of virulence factors in some bacteria and a pause regulating transcription/replication of HIV-1. The results indicate that recognition of RNA-DNA hybrid sequence by multi-subunit RNA polymerases is involved in transcription regulation and may determine the overall rate of transcription elongation.

Authors: Aleksandra Bochkareva, , Vasisht R Tadigotla,

Date Published: 29th Nov 2011

Publication Type: Not specified

Live Cell Imaging of <em>Bacillus subtilis</em> and <em>Streptococcus pneumoniae</em> using Automated Time-lapse Microscopy

Abstract (Expand)

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of … single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see (1,2,3)). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy). Here, we provide a detailed description of a microscopy method used in several recent studies (4, 5, 6, 7), which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Authors: , Katrin Beilharz, ,

Date Published: 16th Aug 2011

Publication Type: Not specified

A new basal promoter element recognized by RNA polymerase core enzyme

Abstract (Expand)

Bacterial promoters are recognized by RNA polymerase (RNAP) σ subunit, which specifically interacts with the -10 and -35 promoter elements. Here, we provide evidence that the β' zipper, an … evolutionarily conserved loop of the largest subunit of RNAP core, interacts with promoter spacer, a DNA segment that separates the -10 and -35 promoter elements, and facilitates the formation of stable closed promoter complex. Depending on the spacer sequence, the proposed interaction of the β' zipper with the spacer can also facilitate open promoter complex formation and even substitute for interactions of the σ subunit with the -35 element. These results suggest that there exists a novel class of promoters that rely on interaction of the β' zipper with promoter spacer, along with or instead of interactions of σ subunit with the -35 element, for their activity. Finally, our data suggest that sequence-dependent interactions of the β' zipper with DNA can contribute to promoter-proximal σ-dependent RNAP pausing, a recently recognized important step of transcription control.

Authors: , Vasisht R Tadigotla, Konstantin Severinov,

Date Published: 26th Jul 2011

Publication Type: Not specified

Nonlinear fitness landscape of a molecular pathway

Abstract (Expand)

Genes are regulated because their expression involves a fitness cost to the organism. The production of proteins by transcription and translation is a well-known cost factor, but the enzymatic activity … of the proteins produced can also reduce fitness, depending on the internal state and the environment of the cell. Here, we map the fitness costs of a key metabolic network, the lactose utilization pathway in Escherichia coli. We measure the growth of several regulatory lac operon mutants in different environments inducing expression of the lac genes. We find a strikingly nonlinear fitness landscape, which depends on the production rate and on the activity rate of the lac proteins. A simple fitness model of the lac pathway, based on elementary biophysical processes, predicts the growth rate of all observed strains. The nonlinearity of fitness is explained by a feedback loop: production and activity of the lac proteins reduce growth, but growth also affects the density of these molecules. This nonlinearity has important consequences for molecular function and evolution. It generates a cliff in the fitness landscape, beyond which populations cannot maintain growth. In viable populations, there is an expression barrier of the lac genes, which cannot be exceeded in any stationary growth process. Furthermore, the nonlinearity determines how the fitness of operon mutants depends on the inducer environment. We argue that fitness nonlinearities, expression barriers, and gene-environment interactions are generic features of fitness landscapes for metabolic pathways, and we discuss their implications for the evolution of regulation.

Authors: Lilia Perfeito, Stéphane Ghozzi, , Karin Schnetz, Michael Lässig

Date Published: 21st Jul 2011

Publication Type: Not specified

SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae

Abstract (Expand)

Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes … is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences (parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δsmc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism.

Authors: Anita Minnen, Laetitia Attaiech, Maria Thon, Stephan Gruber,

Date Published: 22nd Jun 2011

Publication Type: Not specified

Controlled interplay between trigger loop and Gre factor in the RNA polymerase active centre

Abstract (Expand)

The highly processive transcription by multi-subunit RNA polymerases (RNAP) can be interrupted by misincorporation or backtracking events that may stall transcription or lead to erroneous transcripts. … Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript. Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre. This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis. Replacement of the TL by Gre factor occurs only in backtracked/misincorporated complexes, and not in correctly elongating complexes. This controlled switching of RNAP activities allows the processivity of elongation to be unaffected by the hydrolytic activity of Gre, while ensuring efficient proofreading of transcription and resolution of backtracked complexes.

Authors: Mohammad Roghanian, ,

Date Published: 27th Jan 2011

Publication Type: Not specified

DnaA and ORC: more than DNA replication initiators

Abstract (Expand)

Mutations in DNA replication initiator genes in both prokaryotes and eukaryotes lead to a pleiotropic array of phenotypes, including defects in chromosome segregation, cytokinesis, cell cycle regulation … and gene expression. For years, it was not clear whether these diverse effects were indirect consequences of perturbed DNA replication, or whether they indicated that DNA replication initiator proteins had roles beyond their activity in initiating DNA synthesis. Recent work from a range of organisms has demonstrated that DNA replication initiator proteins play direct roles in many cellular processes, often functioning to coordinate the initiation of DNA replication with essential cell-cycle activities. The aim of this review is to highlight these new findings, focusing on the pathways and mechanisms utilized by DNA replication initiator proteins to carry out a diverse array of cellular functions.

Authors: Graham Scholefield, , Heath Murray

Date Published: 27th Aug 2010

Publication Type: Not specified

Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis

Abstract (Expand)

The active center of RNA polymerase can hydrolyze phosphodiester bonds in nascent RNA, a reaction thought to be important for proofreading of transcription. The reaction proceeds via a general two … Mg(2+) mechanism and is assisted by the 3' end nucleotide of the transcript. Here, by using Thermus aquaticus RNA polymerase, we show that the reaction also requires the flexible domain of the active center, the trigger loop (TL). We show that the invariant histidine (beta' His1242) of the TL is essential for hydrolysis/proofreading and participates in the reaction in two distinct ways: by positioning the 3' end nucleotide of the transcript that assists catalysis and/or by directly participating in the reaction as a general base. We also show that participation of the beta' His1242 of the TL in phosphodiester bond hydrolysis does not depend on the extent of elongation complex backtracking. We obtained similar results with Escherichia coli RNA polymerase, indicating that the function of the TL in phosphodiester bond hydrolysis is conserved among bacteria.

Authors: Yulia Yuzenkova,

Date Published: 1st Jun 2010

Publication Type: Not specified

Stepwise mechanism for transcription fidelity

Abstract (Expand)

Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just … non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive.

Authors: , Aleksandra Bochkareva, Vasisht R Tadigotla, Mohammad Roghanian, Savva Zorov, Konstantin Severinov,

Date Published: 1st Apr 2010

Publication Type: Not specified

Gene position within a long transcript as a determinant for stochastic switching in bacteria

Abstract (Expand)

How cultures of genetically identical cells bifurcate into distinct phenotypic subpopulations under uniform growth conditions is an important question in developmental biology of relevance even to … relatively simple developmental systems, such as spore formation in bacteria. A growing Bacillus subtilis culture consists of either cells that are motile and can swim or cells that are non-motile and are chained together. In this issue of Molecular Microbiology, Cozy and Kearns show that the probability of a cell to become motile depends on the position of the sigD gene within the long (27 kb) motility operon. sigD encodes the alternative sigma factor sigma(D) that, together with RNA polymerase, drives expression of genes required for cell separation and the assembly of flagella. sigD is the penultimate gene of the B. subtilis motility operon and, in the control strain approximately, 70% of the cells are motile. When sigD was moved upstream within the operon, a larger fraction of cells became motile (up to 100%). This study highlights that the position of a gene within an operon can have a large impact on the control of gene expression. Furthermore, it suggests that RNA polymerase processivity or mRNA turnover can play important roles as sources of noise in bacterial development, and that gene position might be an unrecognized and possibly widespread mechanism to regulate phenotypic variation.

Editor:

Date Published: 10th Mar 2010

Publication Type: Not specified

Transformation of environmental Bacillus subtilis isolates by transiently inducing genetic competence

Abstract (Expand)

Domesticated laboratory strains of Bacillus subtilis readily take up and integrate exogenous DNA. In contrast, "wild" ancestors or Bacillus strains recently isolated from the environment can … only be genetically modified by phage transduction, electroporation or protoplast transformation. Such methods are laborious, have a variable yield or cannot efficiently be used to alter chromosomal DNA. A major disadvantage of using laboratory strains is that they have often lost, or do not display ecologically relevant physiologies such as the ability to form biofilms. Here we present a method that allows genetic transformation by natural competence in several environmental isolates of B. subtilis. Competence in these strains was established by expressing the B. subtilis competence transcription factor ComK from an IPTG-inducible promoter construct present on an unstable plasmid. This transiently activates expression of the genes required for DNA uptake and recombination in the host strain. After transformation, the comK encoding plasmid is lost easily because of its intrinsic instability and the transformed strain returns to its wild state. Using this method, we have successfully generated mutants and introduced foreign DNA into a number of environmental isolates and also B. subtilis strain NCIB3610, which is widely used to study biofilm formation. Application of the same method to strains of B. licheniformis was unsuccessful. The efficient and rapid approach described here may facilitate genetic studies in a wider array of environmental B. subtilis strains.

Authors: Reindert Nijland, J Grant Burgess, Jeff Errington,

Date Published: 11th Jan 2010

Publication Type: Not specified

Significance analysis and statistical mechanics: an application to clustering

Abstract (Expand)

This Letter addresses the statistical significance of structures in random data: given a set of vectors and a measure of mutual similarity, how likely is it that a subset of these vectors forms a cluster … with enhanced similarity among its elements? The computation of this cluster p value for randomly distributed vectors is mapped onto a well-defined problem of statistical mechanics. We solve this problem analytically, establishing a connection between the physics of quenched disorder and multiple-testing statistics in clustering and related problems. In an application to gene expression data, we find a remarkable link between the statistical significance of a cluster and the functional relationships between its genes.

Authors: Marta Łuksza, Michael Lässig,

Date Published: 27th Nov 2009

Publication Type: Not specified

Cellular localization of choline-utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems

Abstract (Expand)

The molecular mechanisms underlying cell growth, cell division and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to … investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zinc-inducible system for the fine control of gfp-fusion gene expression and for protein depletion experiments in S. pneumoniae. Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.

Authors: Alice Eberhardt, Ling J Wu, Jeff Errington, Waldemar Vollmer,

Date Published: 8th Sep 2009

Publication Type: Not specified

A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis

Abstract (Expand)

Coordination of DNA replication with cellular development is a crucial problem in most living organisms. Bacillus subtilis cells switch from vegetative growth to sporulation when starved. Sporulation … normally occurs in cells that have stopped replicating DNA and have two completed chromosomes: one destined for the prespore and the other for the mother cell. It has long been recognized that there is a sensitive period in the cell cycle during which the initiation of spore development can be triggered, presumably to allow for the generation of exactly two complete chromosomes. However, the mechanism responsible for this has remained unclear. Here we show that the sda gene, previously identified as a checkpoint factor preventing sporulation in response to DNA damage, exerts cell cycle control over the initiation of sporulation. Expression of sda occurs in a pulsatile manner, with a burst of expression each cell cycle at the onset of DNA replication. Up-regulation of the intrinsically unstable Sda protein, which is dependent on the active form of the DNA replication initiator protein, DnaA, transiently inhibits the initiation of sporulation. This regulation avoids the generation of spore formers with replicating chromosomes, which would result in diploid or polyploid spores that we show have reduced viability.

Authors: , Heath Murray, Jeff Errington

Date Published: 18th Aug 2009

Publication Type: Not specified

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