scholarly journals Under Elevated c-di-GMP in Escherichia coli, YcgR Alters Flagellar Motor Bias and Speed Sequentially, with Additional Negative Control of the Flagellar Regulon via the Adaptor Protein RssB

2019 ◽  
Vol 202 (1) ◽  
Author(s):  
Vincent Nieto ◽  
Jonathan D. Partridge ◽  
Geoffrey B. Severin ◽  
Run-Zhi Lai ◽  
Christopher M. Waters ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Negative Control ◽  
Flagellar Motor ◽  
Content Type ◽  
Environmental Signals ◽  
E Coli ◽  
Regulator Protein ◽  
Flagellar Regulon ◽  
Flagellar Assembly ◽  
And Function

Flagellum-driven motility has been studied in E. coli and Salmonella for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in E. coli, we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σS regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated.

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 Diversification of Campylobacter jejuni Flagellar C-Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Louie D. Henderson ◽  
Teige R. S. Matthews-Palmer ◽  
Connor J. Gulbronson ◽  
Deborah A. Ribardo ◽  
Morgan Beeby ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Content Type ◽  
Cellular Processes ◽  
Spatial Regulation ◽  
Flagellar Motors ◽  
Core Components ◽  
Flagellar Assembly ◽  
And Function ◽  
Conserved Core ◽  
Flagellar Biogenesis

ABSTRACT Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species. IMPORTANCE The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Campylobacter jejuni flagellar C ring, the mechanistic core of the motor. Our work provides insight into how usually conserved core components may have diversified by gene duplication, enabling a division of labor of the ancestral protein between the two new proteins, acquisition of new roles in flagellar assembly and motility, and expansion of the function of the flagellum beyond motility, including spatial regulation of cell division and numerical control of flagellar biogenesis in C. jejuni. Our results highlight that relatively small changes, such as gene duplications, can have substantial ramifications on the cellular roles of a molecular machine.

scholarly journals Domain Analysis of the FliM Protein ofEscherichia coli

1998 ◽  
Vol 180 (21) ◽  
pp. 5580-5590 ◽  
Author(s):  
Michael A. A. Mathews ◽  
Hua Lucy Tang ◽  
David F. Blair
Keyword(s):  
Sequence Similarity ◽  
Flagellar Motor ◽  
Binding Studies ◽  
Phosphorylated Protein ◽  
Cell Lysates ◽  
Terminal Domain ◽  
Flagellar Assembly ◽  
Flagellar Proteins ◽  
And Function ◽  
Multiple Domains

ABSTRACT The FliM protein of Escherichia coli is required for the assembly and function of flagella. Genetic analyses and binding studies have shown that FliM interacts with several other flagellar proteins, including FliN, FliG, phosphorylated CheY, other copies of FliM, and possibly MotA and FliF. Here, we examine the effects of a set of linker insertions and partial deletions in FliM on its binding to FliN, FliG, CheY, and phospho-CheY and on its functions in flagellar assembly and rotation. The results suggest that FliM is organized into multiple domains. A C-terminal domain of about 90 residues binds to FliN in coprecipitation experiments, is most stable when coexpressed with FliN, and has some sequence similarity to FliN. This C-terminal domain is joined to the rest of FliM by a segment (residues 237 to 247) that is poorly conserved, tolerates linker insertion, and may be an interdomain linker. Binding to FliG occurs through multiple segments of FliM, some in the C-terminal domain and others in an N-terminal domain of 144 residues. Binding of FliM to CheY and phospho-CheY was complex. In coprecipitation experiments using purified FliM, the protein bound weakly to unphosphorylated CheY and more strongly to phospho-CheY, in agreement with previous reports. By contrast, in experiments using FliM in fresh cell lysates, the protein bound to unphosphorylated CheY about as well as to phospho-CheY. Determinants for binding CheY occur both near the N terminus of FliM, which appears most important for binding to the phosphorylated protein, and in the C-terminal domain, which binds more strongly to unphosphorylated CheY. Several different deletions and linker insertions in FliM enhanced its binding to phospho-CheY in coprecipitation experiments with protein from cell lysates. This suggests that determinants for binding phospho-CheY may be partly masked in the FliM protein as it exists in the cytoplasm. A model is proposed for the arrangement and function of FliM domains in the flagellar motor.

scholarly journals Evidence for Cyclic Di-GMP-Mediated Signaling in Bacillus subtilis

2012 ◽  
Vol 194 (18) ◽  
pp. 5080-5090 ◽  
Author(s):  
Yun Chen ◽  
Yunrong Chai ◽  
Jian-hua Guo ◽  
Richard Losick
Keyword(s):  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Second Messenger ◽  
Negative Control ◽  
Gram Negative Bacteria ◽  
Flagellar Motor ◽  
Positive Bacterium ◽  
Content Type ◽  
Deletion Mutations ◽  
Cellular Processes

ABSTRACTCyclic di-GMP (c-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. Studies of c-di-GMP signaling have chiefly focused on Gram-negative bacteria. Here, we investigated c-di-GMP signaling in the Gram-positive bacteriumBacillus subtilisby constructing deletion mutations in genes predicted to be involved in the synthesis, breakdown, or response to the second messenger. We found that a putative c-di-GMP-degrading phosphodiesterase, YuxH, and a putative c-di-GMP receptor, YpfA, had strong influences on motility and that these effects depended on sequences similar to canonical EAL and RxxxR—D/NxSxxG motifs, respectively. Evidence indicates that YpfA inhibits motility by interacting with the flagellar motor protein MotA and thatyuxHis under the negative control of the master regulator Spo0A∼P. Based on these findings, we propose that YpfA inhibits motility in response to rising levels of c-di-GMP during entry into stationary phase due to the downregulation ofyuxHby Spo0A∼P. We also present evidence that YpfA has a mild influence on biofilm formation.In toto, our results demonstrate the existence of a functional c-di-GMP signaling system inB. subtilisthat directly inhibits motility and directly or indirectly influences biofilm formation.

scholarly journals Requirements for Conversion of the Na+-Driven Flagellar Motor of Vibrio cholerae to the H+-Driven Motor of Escherichia coli

2000 ◽  
Vol 182 (15) ◽  
pp. 4234-4240 ◽  
Author(s):  
Khoosheh K. Gosink ◽  
Claudia C. Häse
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
Proton Translocation ◽  
Specific Inhibitor ◽  
The Other ◽  
E Coli ◽  
Alkaline Conditions ◽  
Flagellar Assembly ◽  
And Function ◽  
Hybrid Motor

ABSTRACT Bacterial flagella are powered by a motor that converts a transmembrane electrochemical potential of either H+ or Na+ into mechanical work. In Escherichia coli, the MotA and MotB proteins form the stator and function in proton translocation, whereas the FliG protein is located on the rotor and is involved in flagellar assembly and torque generation. The sodium-driven polar flagella of Vibrio species contain homologs of MotA and MotB, called PomA and PomB, and also contain two other membrane proteins called MotX and MotY, which are essential for motor rotation and that might also function in ion conduction. Deletions inpomA, pomB, motX, ormotY in Vibrio cholerae resulted in a nonmotile phenotype, whereas deletion of fliG gave a nonflagellate phenotype. fliG genes on plasmids complementedfliG-null strains of the parent species but notfliG-null strains of the other species. FliG-null strains were complemented by chimeric FliG proteins in which the C-terminal domain came from the other species, however, implying that the C-terminal part of FliG can function in conjunction with the ion-translocating components of either species. A V. cholerae strain deleted of pomA, pomB,motX, and motY became weakly motile when theE. coli motA and motB genes were introduced on a plasmid. Like E. coli, but unlike wild-type V. cholerae, motility of some V. cholerae strains containing the hybrid motor was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone under neutral as well as alkaline conditions but not by the sodium motor-specific inhibitor phenamil. We conclude that the E. coli proton motor components MotA and MotB can function in place of the motor proteins ofV. cholerae and that the hybrid motors are driven by the proton motive force.

scholarly journals The Intimin-Like Protein FdeC Is Regulated by H-NS and Temperature in Enterohemorrhagic Escherichia coli

2014 ◽  
Vol 80 (23) ◽  
pp. 7337-7347 ◽  
Author(s):  
Donna M. Easton ◽  
Luke P. Allsopp ◽  
Minh-Duy Phan ◽  
Danilo Gomes Moriel ◽  
Guan Kai Goh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Culture Conditions ◽  
Enterohemorrhagic Escherichia Coli ◽  
Infection Model ◽  
Content Type ◽  
E Coli ◽  
Uremic Syndrome ◽  
Animal Hosts ◽  
And Function

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) is a Shiga-toxigenic pathogen capable of inducing severe forms of enteritis (e.g., hemorrhagic colitis) and extraintestinal sequelae (e.g., hemolytic-uremic syndrome). The molecular basis of colonization of human and animal hosts by EHEC is not yet completely understood, and an improved understanding of EHEC mucosal adherence may lead to the development of interventions that could disrupt host colonization. FdeC, also referred to by its IHE3034 locus tag ECOK1_0290, is an intimin-like protein that was recently shown to contribute to kidney colonization in a mouse urinary tract infection model. The expression of FdeC is tightly regulatedin vitro, and FdeC shows promise as a vaccine candidate against extraintestinalE. colistrains. In this study, we characterized the prevalence, regulation, and function offdeCin EHEC. We showed that thefdeCgene is conserved in both O157 and non-O157 EHEC and encodes a protein that is expressed at the cell surface and promotes biofilm formation under continuous-flow conditions in a recombinantE. colistrain background. We also identified culture conditions under which FdeC is expressed and showed that minor alterations of these conditions, such as changes in temperature, can significantly alter the level of FdeC expression. Additionally, we demonstrated that the transcription of thefdeCgene is repressed by the global regulator H-NS. Taken together, our data suggest a role for FdeC in EHEC when it grows at temperatures above 37°C, a condition relevant to its specialized niche at the rectoanal junctions of cattle.

scholarly journals Pseudouridine-FreeEscherichia coliRibosomes

2017 ◽  
Vol 200 (4) ◽  
Author(s):  
Michael O'Connor ◽  
Margus Leppik ◽  
Jaanus Remme
Keyword(s):  
Cell Growth ◽  
Functional Role ◽  
Ribosome Biogenesis ◽  
Content Type ◽  
E Coli ◽  
Pseudouridine Synthase ◽  
Domains Of Life ◽  
And Function ◽  
Critical Regions ◽  
Dependent Mechanism

ABSTRACTPseudouridine (Ψ) is present at conserved, functionally important regions in the ribosomal RNAs (rRNAs) from all three domains of life. Little, however, is known about the functions of Ψ modifications in bacterial ribosomes. AnEscherichia colistrain has been constructed in which all seven rRNA Ψ synthases have been inactivated and whose ribosomes are devoid of all Ψs. Surprisingly, this strain displays only minor defects in ribosome biogenesis and function, and cell growth is only modestly affected. This is in contrast to a strong requirement for Ψ in eukaryotic ribosomes and suggests divergent roles for rRNA Ψ modifications in these two domains.IMPORTANCEPseudouridine (Ψ) is the most abundant posttranscriptional modification in RNAs. In the ribosome, Ψ modifications are typically located at conserved, critical regions, suggesting they play an important functional role. In eukarya and archaea, rRNAs are modified by a single pseudouridine synthase (PUS) enzyme, targeted to rRNA via a snoRNA-dependent mechanism, while bacteria use multiple stand-alone PUS enzymes. Disruption of Ψ modification of rRNA in eukarya seriously impairs ribosome function and cell growth. We have constructed anE. colimultiple deletion strain lacking all Ψ modifications in rRNA. In contrast to the equivalent eukaryotic mutants, theE. colistrain is only modestly affected in growth, decoding, and ribosome biogenesis, indicating a differential requirement for Ψ modifications in these two domains.

scholarly journals Integrated Regulation of Acetoin Fermentation by Quorum Sensing and pH in Serratia plymuthica RVH1

2011 ◽  
Vol 77 (10) ◽  
pp. 3422-3427 ◽  
Author(s):  
Pieter Moons ◽  
Rob Van Houdt ◽  
Bram Vivijs ◽  
Chris M. Michiels ◽  
Abram Aertsen
Keyword(s):  
Quorum Sensing ◽  
Bacterial Species ◽  
Homoserine Lactone ◽  
Positive Control ◽  
Negative Control ◽  
Serratia Plymuthica ◽  
Content Type ◽  
Environmental Signals ◽  
Mixed Acid ◽  
External Stimuli

ABSTRACTDuring fermentation of sugars, a number of bacterial species are able to switch from mixed acid production to acetoin and 2,3-butanediol production in order to avoid lethal acidification of their environment, although the regulation of this switch is only poorly understood. In this study, we report the identification of thebudABstructural operon, involved in acetoin production inSerratia plymuthicaRVH1, and its activation by a LysR-type regulator encoded bybudR, immediately upstream of this operon. In addition, the regulation ofbudRtranscription was elucidated and found to be subject to negative control by BudR itself and to positive control by external stimuli such asN-(3-oxohexanoyl)-l-homoserine lactone (OHHL) quorum sensing signaling molecules and acetate. Interestingly, however, we observed that induction ofbudRtranscription by OHHL or acetate did not require BudR, indicating the involvement of additional regulatory factors in relaying these environmental signals to thebudRpromoter.

scholarly journals Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Inflammation and Cell Damage via Attenuation of ASC-Independent NLRP3 Inflammasome Activation

2015 ◽  
Vol 82 (4) ◽  
pp. 1173-1182 ◽  
Author(s):  
Qiong Wu ◽  
Ming-Chao Liu ◽  
Jun Yang ◽  
Jiu-Feng Wang ◽  
Yao-Hong Zhu
Keyword(s):  
Escherichia Coli ◽  
Mrna Expression ◽  
Nlrp3 Inflammasome ◽  
Cell Damage ◽  
Lactobacillus Rhamnosus ◽  
Adaptor Protein ◽  
Inflammasome Activation ◽  
Content Type ◽  
E Coli ◽  
Nlrp3 Inflammasome Activation

ABSTRACTEscherichia coliis a major environmental pathogen causing bovine mastitis, which leads to mammary tissue damage and cell death. We explored the effects of the probioticLactobacillus rhamnosusGR-1 on amelioratingE. coli-induced inflammation and cell damage in primary bovine mammary epithelial cells (BMECs). Increased Toll-like receptor 4 (TLR4), NOD1, and NOD2 mRNA expression was observed followingE. colichallenge, but this increase was attenuated byL. rhamnosusGR-1 pretreatment. Immunofluorescence and Western blot analyses revealed thatL. rhamnosusGR-1 pretreatment decreased theE. coli-induced increases in the expression of the NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) and the serine protease caspase 1. However, expression of the adaptor protein apoptosis-associated speck-like protein (ASC, encoded by thePycardgene) was decreased duringE. coliinfection, even withL. rhamnosusGR-1 pretreatment. Pretreatment withL. rhamnosusGR-1 counteracted theE. coli-induced increases in interleukin-1β (IL-1β), -6, -8, and -18 and tumor necrosis factor alpha mRNA expression but upregulated IL-10 mRNA expression. Our data indicate thatL. rhamnosusGR-1 reduces the adhesion ofE. colito BMECs, subsequently amelioratingE. coli-induced disruption of cellular morphology and ultrastructure and limiting detrimental inflammatory responses, partly via promoting TLR2 and NOD1 synergism and attenuating ASC-independent NLRP3 inflammasome activation. Although the residual pathogenic activity ofL. rhamnosus, the dosage regimen, and the means of probiotic supplementation in cattle remain undefined, our data enhance our understanding of the mechanism of action of this candidate probiotic, allowing for development of specific probiotic-based therapies and strategies for preventing pathogenic infection of the bovine mammary gland.

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