scholarly journals Dynamics of the Bacillus subtilis Min System

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Helge Feddersen ◽  
Laeschkir Würthner ◽  
Erwin Frey ◽  
Marc Bramkamp
Keyword(s):  
Bacillus Subtilis ◽  
Protein Dynamics ◽  
Site Selection ◽  
Active Sites ◽  
Molecular Mechanisms ◽  
Content Type ◽  
E Coli ◽  
Protein System ◽  
Division Site ◽  
Min Proteins

ABSTRACT Division site selection is a vital process to ensure generation of viable offspring. In many rod-shaped bacteria, a dynamic protein system, termed the Min system, acts as a central regulator of division site placement. The Min system is best studied in Escherichia coli, where it shows a remarkable oscillation from pole to pole with a time-averaged density minimum at midcell. Several components of the Min system are conserved in the Gram-positive model organism Bacillus subtilis. However, in B. subtilis, it is commonly believed that the system forms a stationary bipolar gradient from the cell poles to midcell. Here, we show that the Min system of B. subtilis localizes dynamically to active sites of division, often organized in clusters. We provide physical modeling using measured diffusion constants that describe the observed enrichment of the Min system at the septum. Mathematical modeling suggests that the observed localization pattern of Min proteins corresponds to a dynamic equilibrium state. Our data provide evidence for the importance of ongoing septation for the Min dynamics, consistent with a major role of the Min system in controlling active division sites but not cell pole areas. IMPORTANCE The molecular mechanisms that help to place the division septum in bacteria is of fundamental importance to ensure cell proliferation and maintenance of cell shape and size. The Min protein system, found in many rod-shaped bacteria, is thought to play a major role in division site selection. It was assumed that there are strong differences in the functioning and in the dynamics of the Min system in E. coli and B. subtilis. Most previous attempts to address Min protein dynamics in B. subtilis have been hampered by the use of overexpression constructs. Here, functional fusions to Min proteins have been constructed by allelic exchange and state-of-the-art imaging techniques allowed to unravel an unexpected fast dynamic behavior of the B. subtilis Min system. Our data show that the molecular mechanisms leading to Min protein dynamics are not fundamentally different in E. coli and B. subtilis.

scholarly journals Dynamics of the Bacillus subtilis Min system

2020 ◽  
Author(s):  
Helge Feddersen ◽  
Laeschkir Würthner ◽  
Erwin Frey ◽  
Marc Bramkamp
Keyword(s):  
Bacillus Subtilis ◽  
Active Sites ◽  
Dynamic Equilibrium ◽  
Model Organism ◽  
Physical Modelling ◽  
Division Site ◽  
Diffusion Constants ◽  
Dynamic Equilibrium State ◽  
Min Proteins

SummaryDivision site selection is a vital process to ensure generation of viable offspring. In many rod-shaped bacteria a dynamic protein system, termed the Min system, acts as a central regulator of division site placement. The Min system is best studied in Escherichia coli where it shows a remarkable oscillation from pole to pole with a time-averaged density minimum at midcell. Several components of the Min system are conserved in the Gram-positive model organism Bacillus subtilis. However, in B. subtilis it is believed that the system forms a stationary bipolar gradient from the cell poles to midcell. Here, we show that the Min system of B. subtilis localizes dynamically to active sites of division, often organized in clusters. We provide physical modelling using measured diffusion constants that describe the observed enrichment of the Min system at the septum. Modelling suggests that the observed localization pattern of Min proteins corresponds to a dynamic equilibrium state. Our data provide evidence for the importance of ongoing septation for the Min dynamics, consistent with a major role of the Min system to control active division sites, but not cell pole areas.

scholarly journals Organization of the Flagellar Switch Complex ofBacillus subtilis

2018 ◽  
Vol 201 (8) ◽  
Author(s):  
Elizabeth Ward ◽  
Eun A Kim ◽  
Joseph Panushka ◽  
Tayson Botelho ◽  
Trevor Meyer ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interaction ◽  
Protein Complex ◽  
Cross Linking ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Middle Domain ◽  
Flagellar Rotation

ABSTRACTWhile the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied inEscherichia coliandSalmonella, less is known about the switch complex inBacillus subtilisor other Gram-positive species. Two component proteins (FliG and FliM) are shared betweenE. coliandB. subtilis, but in place of the protein FliN found inE. coli, theB. subtiliscomplex contains the larger protein FliY. Notably, inB. subtilisthe signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action inE. coli. Here, we have examined the architecture and function of the switch complex inB. subtilisusing targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, theB. subtilisswitch complex appears to be organized similarly to that inE. coli. The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that ofE. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences fromE. coliinvolve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCEFlagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex inBacillus subtilisor other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas inE. coliorSalmonellaCheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of theB. subtilisswitch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control inB. subtilisand other Gram-positive species.

scholarly journals Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Charles T. Lauhon
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Genomic Context ◽  
Similar Reduction ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Fertile Ground ◽  
And Function

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

scholarly journals Novel Quorum-Sensing Peptides Mediating Interspecies Bacterial Cell Death

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Sathish Kumar ◽  
Ilana Kolodkin-Gal ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Bacillus Subtilis ◽  
Quorum Sensing ◽  
Bacterial Cell ◽  
Content Type ◽  
E Coli ◽  
New Class ◽  
Bacterial Cell Death ◽  
Induced Module

ABSTRACTEscherichia colimazEFis a toxin-antitoxin stress-induced module mediating cell death. It requires the quorum-sensing signal (QS) “extracellular death factor” (EDF), the penta-peptide NNWNN (EcEDF), enhancing the endoribonucleolytic activity ofE. colitoxin MazF. Here we discovered thatE. coli mazEF-mediated cell death could be triggered by QS peptides from the supernatants (SN) of the Gram-positive bacteriumBacillus subtilisand the Gram-negative bacteriumPseudomonas aeruginosa. In the SN ofB. subtilis, we found one EDF, the hexapeptide RGQQNE, calledBsEDF. In the SN ofP. aeruginosa, we found three EDFs: the nonapeptide INEQTVVTK, calledPaEDF-1, and two hexadecapeptides, VEVSDDGSGGNTSLSQ, calledPaEDF-2, and APKLSDGAAAGYVTKA, calledPaEDF-3. When added to a dilutedE. colicultures, each of these peptides acted as an interspecies EDF that triggeredmazEF-mediated death. Furthermore, though their sequences are very different, each of these EDFs amplified the endoribonucleolytic activity ofE. coliMazF, probably by interacting with different sites onE. coliMazF. Finally, we suggest that EDFs may become the basis for a new class of antibiotics that trigger death from outside the bacterial cells.IMPORTANCEBacteria communicate with one another via quorum-sensing signal (QS) molecules. QS provides a mechanism for bacteria to monitor each other’s presence and to modulate gene expression in response to population density. Previously, we addedE. coliEDF (EcEDF), the peptide NNWNN, to this list of QS molecules. Here we extended the group of QS peptides to several additional different peptides. The new EDFs are produced by two other bacteria,Bacillus subtilisandPseudomonas aeruginosa. Thus, in this study we established a “new family of EDFs.” This family provides the first example of quorum-sensing molecules participating in interspecies bacterial cell death. Furthermore, each of these peptides provides the basis of a new class of antibiotics triggering death by acting from outside the cell.

scholarly journals Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

2011 ◽  
Vol 77 (20) ◽  
pp. 7104-7112 ◽  
Author(s):  
Maria Karczmarczyk ◽  
Yvonne Abbott ◽  
Ciara Walsh ◽  
Nola Leonard ◽  
Séamus Fanning
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Gene Array ◽  
Multidrug Resistant ◽  
Genetic Determinants ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Class 1

ABSTRACTIn this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection ofEscherichia coliisolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1,dfrA1-aadA1,dfrA17-aadA5,dfrA12-orfF-aadA2,blaOXA-30-aadA1,aacC1-orf1-orf2-aadA1,dfr7). Class 2 integrons (13.5%) contained thedfrA1-sat1-aadA1gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected includedblaTEM,cat,floR,aadB,aphA1,strA-strB,sul2, andtet(B), respectively. TheblaCTX-M-2gene, encoding an extended-spectrum β-lactamase (ESβL), andblaCMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensalE. coliisolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, theblaCTX-M-2gene has not previously been reported in Ireland.

scholarly journals Transcriptional Responses of Escherichia coli K-12 and O157:H7 Associated with Lettuce Leaves

2012 ◽  
Vol 78 (6) ◽  
pp. 1752-1764 ◽  
Author(s):  
Ryan C. Fink ◽  
Elaine P. Black ◽  
Zhe Hou ◽  
Masayuki Sugawara ◽  
Michael J. Sadowsky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Leaf Surface ◽  
Short Term ◽  
Transcriptional Responses ◽  
Content Type ◽  
E Coli ◽  
Leaf Surfaces ◽  
K 12

ABSTRACTAn increasing number of outbreaks of gastroenteritis recently caused byEscherichia coliO157:H7 have been linked to the consumption of leafy green vegetables. Although it is known thatE. colisurvives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identifyE. coligenes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparingE. coliK-12, a model system, andE. coliO157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, includingtnaA(33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsAandybiM) and curli production (csgAandcsgB) were significantly upregulated inE. coliK-12 and O157:H7. BothcsgAandbhsA(ycfR) mutants were impaired in the long-term colonization of the leaf surface, but onlycsgAmutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction ofE. coliK-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation.

scholarly journals Role of F1C Fimbriae, Flagella, and Secreted Bacterial Components in the Inhibitory Effect of Probiotic Escherichia coli Nissle 1917 on Atypical Enteropathogenic E. coli Infection

2014 ◽  
Vol 82 (5) ◽  
pp. 1801-1812 ◽  
Author(s):  
Sylvia Kleta ◽  
Marcel Nordhoff ◽  
Karsten Tedin ◽  
Lothar H. Wieler ◽  
Rafal Kolenda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Inhibitory Effect ◽  
Host Cells ◽  
Single Infection ◽  
Content Type ◽  
E Coli ◽  
Initial Adhesion

ABSTRACTEnteropathogenicEscherichia coli(EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probioticE. colistrain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While bothin vitroandin vivostudies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenicE. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenicE. coli(aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression.

scholarly journals Genetic Dissection of DivIVA Functions in Listeria monocytogenes

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
Karan Gautam Kaval ◽  
Samuel Hauf ◽  
Jeanine Rismondo ◽  
Birgitt Hahn ◽  
Sven Halbedel
Keyword(s):  
Cell Division ◽  
Listeria Monocytogenes ◽  
Protein Interactions ◽  
Site Selection ◽  
Genetic Dissection ◽  
Flagellar Motility ◽  
Content Type ◽  
Division Site ◽  
Interaction Partners ◽  
Species Specific

ABSTRACT DivIVA is a membrane binding protein that clusters at curved membrane regions, such as the cell poles and the membrane invaginations occurring during cell division. DivIVA proteins recruit many other proteins to these subcellular sites through direct protein-protein interactions. DivIVA-dependent functions are typically associated with cell growth and division, even though species-specific differences in the spectrum of DivIVA functions and their causative interaction partners exist. DivIVA from the Gram-positive human pathogen Listeria monocytogenes has at least three different functions. In this bacterium, DivIVA is required for precise positioning of the septum at midcell, it contributes to the secretion of autolysins required for the breakdown of peptidoglycan at the septum after the completion of cell division, and it is essential for flagellar motility. While the DivIVA interaction partners for control of division site selection are well established, the proteins connecting DivIVA with autolysin secretion or swarming motility are completely unknown. We set out to identify divIVA alleles in which these three DivIVA functions could be separated, since the question of the degree to which the three functions of L. monocytogenes DivIVA are interlinked could not be answered before. Here, we identify such alleles, and our results show that division site selection, autolysin secretion, and swarming represent three discrete pathways that are independently influenced by DivIVA. These findings provide the required basis for the identification of DivIVA interaction partners controlling autolysin secretion and swarming in the future. IMPORTANCE DivIVA of the pathogenic bacterium Listeria monocytogenes is a central scaffold protein that influences at least three different cellular processes, namely, cell division, protein secretion, and bacterial motility. How DivIVA coordinates these rather unrelated processes is not known. We here identify variants of L. monocytogenes DivIVA, in which these functions are separated from each other. These results have important implications for the models explaining how DivIVA interacts with other proteins.

scholarly journals Pirated Siderophores Promote Sporulation in Bacillus subtilis

2017 ◽  
Vol 83 (10) ◽  
Author(s):  
Gabrielle M. Grandchamp ◽  
Lews Caro ◽  
Elizabeth A. Shank
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Iron Acquisition ◽  
Microbial Interactions ◽  
Mineral Nutrient ◽  
Cell Physiology ◽  
Content Type ◽  
E Coli ◽  
Esterase Gene

ABSTRACT In microbial communities, bacteria chemically and physically interact with one another. Some of these interactions are mediated by secreted specialized metabolites that act as either intraspecies or interspecies signals to alter gene expression and to change cell physiology. Bacillus subtilis is a well-characterized soil microbe that can differentiate into multiple cell types, including metabolically dormant endospores. We were interested in identifying microbial interactions that affected sporulation in B. subtilis. Using a fluorescent transcriptional reporter, we observed that coculturing B. subtilis with Escherichia coli promoted sporulation gene expression via a secreted metabolite. To identify the active compound, we screened the E. coli Keio Collection and identified the sporulation-accelerating cue as the siderophore enterobactin. B. subtilis has multiple iron acquisition systems that are used to take up the B. subtilis-produced siderophore bacillibactin, as well as to pirate exogenous siderophores such as enterobactin. While B. subtilis uses a single substrate binding protein (FeuA) to take up both bacillibactin and enterobactin, we discovered that it requires two distinct genes to sporulate in response to these siderophores (the esterase gene besA for bacillibactin and a putative esterase gene, ybbA, for enterobactin). In addition, we found that siderophores from a variety of other microbial species also promote sporulation in B. subtilis. Our results thus demonstrate that siderophores can act not only as bacterial iron acquisition systems but also as interspecies cues that alter cellular development and accelerate sporulation in B. subtilis. IMPORTANCE While much is known about the genetic regulation of Bacillus subtilis sporulation, little is understood about how other bacteria influence this process. This work describes an interaction between Escherichia coli and B. subtilis that accelerates sporulation in B. subtilis. The interaction is mediated by the E. coli siderophore enterobactin; we show that other species' siderophores also promote sporulation gene expression in B. subtilis. These results suggest that siderophores not only may supply bacteria with the mineral nutrient iron but also may play a role in bacterial interspecies signaling, providing a cue for sporulation. Siderophores are produced by many bacterial species and thus potentially play important roles in altering bacterial cell physiology in diverse environments.

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