scholarly journals Identification of Novel Spx Regulatory Pathways inBacillus subtilisUncovers a Close Relationship between the CtsR and Spx Regulons

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Daniel F. Rojas-Tapias ◽  
John D. Helmann
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Arginine Kinase ◽  
Feedback Mechanism ◽  
Protein Stabilization ◽  
Dual Function ◽  
Content Type ◽  
Regulatory Pathways

ABSTRACTInBacillus subtilis, the Spx transcription factor controls a large regulon in response to disulfide, heat, and cell wall stresses. The regulatory mechanisms that activate the Spx regulon are remarkably complex and involve changes in transcription, proteolysis, and posttranslational modifications. To identify genes involved in Spx regulation, we performed a transposon screen for mutations affecting expression oftrxB, an Spx-dependent gene. Inactivation ofctsR, encoding the regulator of the Clp proteases, reducedtrxBexpression and lowered Spx levels. This effect required ClpP, but involved ClpC rather than the ClpX unfoldase. Moreover, cells lacking McsB, a dual function arginine kinase and ClpCP adaptor, largely reverted thectsRphenotype and increasedtrxBexpression. The role of McsB appears to involve its kinase activity, since loss of the YwlE phosphoarginine phosphatase also led to reducedtrxBexpression. Finally, we show that Spx is itself a regulator of thectsRoperon. Altogether, this work provides evidence for a role of CtsR regulon members ClpC, ClpP, and McsB in Spx regulation and identifies a new feedback pathway associated with Spx activity inB. subtilis.IMPORTANCEInBacillus subtilis, the Spx transcription factor is proteolytically unstable, and protein stabilization figures prominently in the induction of the Spx regulon in response to oxidative and cell envelope stresses. ClpXP is largely, but not entirely, responsible for Spx instability. Here, we identify ClpCP as the protease that degrades Spx under conditions that antagonize the ClpXP pathway. Spx itself contributes to activation of thectsRoperon, which encodes ClpC as well as the McsB arginine kinase and protease adaptor, thereby providing a negative feedback mechanism. Genetic studies reveal that dysregulation of the CtsR regulon or inactivation of the YwlE phosphoarginine phosphatase decreases Spx activity through mechanisms involving both protein degradation and posttranslational modification.

scholarly journals Moonlighting in Bacillus subtilis: The Small Proteins SR1P and SR7P Regulate the Moonlighting Activity of Glyceraldehyde 3-Phosphate Dehydrogenase A (GapA) and Enolase in RNA Degradation

2021 ◽  
Vol 9 (5) ◽  
pp. 1046
Author(s):  
Inam Ul Haq ◽  
Sabine Brantl
Keyword(s):  
Bacillus Subtilis ◽  
Antisense Rna ◽  
Rna Binding ◽  
Rna Degradation ◽  
Metabolic Enzyme ◽  
Dual Function ◽  
Small Proteins ◽  
Moonlighting Proteins ◽  
Rnase Y

Moonlighting proteins are proteins with more than one function. During the past 25 years, they have been found to be rather widespread in bacteria. In Bacillus subtilis, moonlighting has been disclosed to occur via DNA, protein or RNA binding or protein phosphorylation. In addition, two metabolic enzymes, enolase and phosphofructokinase, were localized in the degradosome-like network (DLN) where they were thought to be scaffolding components. The DLN comprises the major endoribonuclease RNase Y, 3′-5′ exoribonuclease PnpA, endo/5′-3′ exoribonucleases J1/J2 and helicase CshA. We have ascertained that the metabolic enzyme GapA is an additional component of the DLN. In addition, we identified two small proteins that bind scaffolding components of the degradosome: SR1P encoded by the dual-function sRNA SR1 binds GapA, promotes the GapA-RNase J1 interaction and increases the RNase J1 activity. SR7P encoded by the dual-function antisense RNA SR7 binds to enolase thereby enhancing the enzymatic activity of enolase bound RNase Y. We discuss the role of small proteins in modulating the activity of two moonlighting proteins.

scholarly journals Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

2016 ◽  
Vol 198 (21) ◽  
pp. 2925-2935 ◽  
Author(s):  
Heng Zhao ◽  
Yingjie Sun ◽  
Jason M. Peters ◽  
Carol A. Gross ◽  
Ethan C. Garner ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Repression ◽  
Cell Envelope ◽  
Turgor Pressure ◽  
Teichoic Acid ◽  
Lethal Gene ◽  
Genetic Redundancy ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Lipid Ii

ABSTRACTThe integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. InBacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C55lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimizedclusteredregularlyinterspacedshortpalindromicrepeat (CRISPR) system with catalytically inactive (“dead”)CRISPR-associated protein9(dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate thatB. subtilisrequires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the σM-dependent cell envelope stress response, includingbcrC, which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of theB. subtilisUPP-Pase enzymes, and provide further evidence linking the σMregulon to cell envelope homeostasis pathways.IMPORTANCEThe emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

scholarly journals Insights into the role of lipoteichoic acids in Bacillus subtilis- a new function for MprF

2021 ◽  
Author(s):  
Aurelie Guyet ◽  
Amirah Alofi ◽  
Richard A Daniel
Keyword(s):  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Cellular Level ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Peptidoglycan Synthesis ◽  
A Cell ◽  
Antimicrobial Peptide Resistance ◽  
Lipoteichoic Acids

In Bacillus subtilis, the cell is protected from the environment by a cell envelope, which comprises of layers of peptidoglycan that maintain the cell shape and anionic teichoic acids polymers whose biological function remains unclear. In B. subtilis, loss of all Class A Penicillin-Binding Proteins (aPBPs) which function in peptidoglycan synthesis is conditionally lethal. Here we show that this lethality is associated with an alteration of the lipoteichoic acids (LTA) and the accumulation of the major autolysin LytE in the cell wall. We provide the first evidence that the length and abundance of LTA acts to regulate the cellular level of LytE. Importantly, we identify a novel function for the aminoacyl-phosphatidylglycerol synthase MprF which acts to modulate LTA biosynthesis in B. subtilis and in the pathogen Staphylococcus aureus. This finding has implications for our understanding of antimicrobial peptide resistance (particularly daptomycin) in clinically relevant bacteria and MprF-associated virulence in pathogens, such as methicillin resistant S. aureus.

scholarly journals Molecular Basis of Substrate Recognition of Endonuclease Q from the Euryarchaeon Pyrococcus furiosus

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Miyako Shiraishi ◽  
Shigenori Iwai
Keyword(s):  
Dna Repair ◽  
Bacillus Subtilis ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Pyrococcus Furiosus ◽  
Substrate Recognition ◽  
Base Flipping ◽  
Content Type ◽  
Cleavage Activity

ABSTRACT Endonuclease Q (EndoQ), a DNA repair endonuclease, was originally identified in the hyperthermophilic euryarchaeon Pyrococcus furiosus in 2015. EndoQ initiates DNA repair by generating a nick on DNA strands containing deaminated bases and an abasic site. Although EndoQ is thought to be important for maintaining genome integrity in certain bacteria and archaea, the underlying mechanism catalyzed by EndoQ remains unclear. Here, we provide insights into the molecular basis of substrate recognition by EndoQ from P. furiosus (PfuEndoQ) using biochemical approaches. Our results of the substrate specificity range and the kinetic properties of PfuEndoQ demonstrate that PfuEndoQ prefers the imide structure in nucleobases along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. The combined results for EndoQ substrate binding and cleavage activity analyses indicated that PfuEndoQ flips the target base from the DNA duplex, and the cleavage activity is highly dependent on spontaneous base flipping of the target base. Furthermore, we find that PfuEndoQ has a relatively relaxed substrate specificity; therefore, the role of EndoQ in restriction modification systems was explored. The activity of the EndoQ homolog from Bacillus subtilis was found not to be inhibited by the uracil glycosylase inhibitor from B. subtilis bacteriophage PBS1, whose genome is completely replaced by uracil instead of thymine. Our findings suggest that EndoQ not only has additional functions in DNA repair but also could act as an antiviral enzyme in organisms with EndoQ. IMPORTANCE Endonuclease Q (EndoQ) is a lesion-specific DNA repair enzyme present in certain bacteria and archaea. To date, it remains unclear how EndoQ recognizes damaged bases. Understanding the mechanism of substrate recognition by EndoQ is important to grasp genome maintenance systems in organisms with EndoQ. Here, we find that EndoQ from the euryarchaeon Pyrococcus furiosus recognizes the imide structure in nucleobases by base flipping, and the cleavage activity is enhanced by the base pair instability of the target base, along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. Furthermore, a potential role of EndoQ in Bacillus subtilis as an antiviral enzyme by digesting viral genome is demonstrated.

scholarly journals Role of SpoIVA ATPase Motifs during Clostridioides difficile Sporulation

2020 ◽  
Vol 202 (21) ◽  
Author(s):  
Hector Benito de la Puebla ◽  
David Giacalone ◽  
Alexei Cooper ◽  
Aimee Shen
Keyword(s):  
Bacillus Subtilis ◽  
Atp Hydrolysis ◽  
Spore Formation ◽  
Spore Form ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Allele Specific ◽  
Coat Layer ◽  
Quality Control Mechanism

ABSTRACT The nosocomial pathogen Clostridioides difficile is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein SpoIVA and the clostridial-organism-specific spore protein SipL, which directly interact. Mutations that disrupt their interaction cause the coat to mislocalize and impair spore formation. In Bacillus subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in the C. difficile SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104-fold less severe than the equivalent mutations in B. subtilis. Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased the SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced the SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele specific, our data imply that SipL recognizes the ATP-bound form of SpoIVA and highlight the importance of this interaction for functional C. difficile spore formation. IMPORTANCE The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

scholarly journals Microbial Gutta-Percha Degradation Shares Common Steps with Rubber Degradation by Nocardia nova SH22a

2012 ◽  
Vol 79 (4) ◽  
pp. 1140-1149 ◽  
Author(s):  
Quan Luo ◽  
Sebastian Hiessl ◽  
Anja Poehlein ◽  
Alexander Steinbüchel
Keyword(s):  
Plasmid Dna ◽  
Cell Envelope ◽  
Degradation Mechanism ◽  
Degradation Pathways ◽  
Genetic Studies ◽  
Content Type ◽  
Gutta Percha ◽  
Optimized Protocol ◽  
Rubber Degradation

ABSTRACTNocardia novaSH22a, a bacterium capable of degrading gutta-percha (GP) and natural rubber (NR), was used to investigate the GP degradation mechanism and the relations between the GP and NR degradation pathways. For this strain, a protocol of electroporation was systematically optimized, and an efficiency of up to 4.3 × 107CFU per μg of plasmid DNA was achieved. By applying this optimized protocol toN. novaSH22a, a Tn5096-based transposon mutagenesis library of this bacterium was constructed. Among about 12,000 apramycin-resistant transformants, we identified 76 stable mutants defective in GP or NR utilization. Whereas 10 mutants were specifically defective in GP utilization, the growth of the other 66 mutants was affected on both GP and NR. This indicated that the two degradation pathways are quite similar and share many common steps. The larger number of GP-degrading defective mutants could be explained in one of two ways: either (i) the GP pathway is more complex and harbors more specific steps or (ii) the steps for both pathways are almost identical, but in the case of GP degradation there are fewer enzymes involved in each step. The analysis of transposition loci and genetic studies on interesting genes confirmed the crucial role of an α-methylacyl-coenzyme A racemase in the degradation of both GP and NR. We also demonstrated the probable involvement of enzymes participating in oxidoreduction reactions, β-oxidation, and the synthesis of complex cell envelope lipids in the degradation of GP.

scholarly journals The Global Transcription Factor Lrp Controls Virulence Modulation in Xenorhabdus nematophila

2015 ◽  
Vol 197 (18) ◽  
pp. 3015-3025 ◽  
Author(s):  
Elizabeth A. Hussa ◽  
Ángel M. Casanova-Torres ◽  
Heidi Goodrich-Blair
Keyword(s):  
Transcription Factor ◽  
Phenotypic Variation ◽  
Pathogenic Bacteria ◽  
Regulatory Protein ◽  
Inverse Correlation ◽  
Xenorhabdus Nematophila ◽  
Insect Larvae ◽  
Content Type ◽  
Expression Levels

ABSTRACTThe bacteriumXenorhabdus nematophilaengages in phenotypic variation with respect to pathogenicity against insect larvae, yielding both virulent and attenuated subpopulations of cells from an isogenic culture. The global regulatory protein Lrp is necessary forX. nematophilavirulence and immunosuppression in insects, as well as colonization of the mutualistic host nematodeSteinernema carpocapsae, and mediates expression of numerous genes implicated in each of these phenotypes. Given the central role of Lrp inX. nematophilahost associations, as well as its involvement in regulating phenotypic variation pathways in other bacteria, we assessed its function in virulence modulation. We discovered that expression oflrpvaries within an isogenic population, in a manner that correlates with modulation of virulence. Unexpectedly, although Lrp is necessary for optimal virulence and immunosuppression, cells expressing high levels oflrpwere attenuated in these processes relative to those with low to intermediatelrpexpression. Furthermore, fixed expression oflrpat high and low levels resulted in attenuated and normal virulence and immunosuppression, respectively, and eliminated population variability of these phenotypes. These data suggest that fluctuatinglrpexpression levels are sufficient to drive phenotypic variation inX. nematophila.IMPORTANCEMany bacteria use cell-to-cell phenotypic variation, characterized by distinct phenotypic subpopulations within an isogenic population, to cope with environmental change. Pathogenic bacteria utilize this strategy to vary antigen or virulence factor expression. Our work establishes that the global transcription factor Lrp regulates phenotypic variation in the insect pathogenXenorhabdus nematophila, leading to attenuation of virulence and immunosuppression in insect hosts. Unexpectedly, we found an inverse correlation between Lrp expression levels and virulence: high levels of expression of Lrp-dependent putative virulence genes are detrimental for virulence but may have an adaptive advantage in other aspects of the life cycle. Investigation ofX. nematophilaphenotypic variation facilitates dissection of this phenomenon in the context of a naturally occurring symbiosis.

scholarly journals Contrasting Effects of σE on Compartmentalization of σF Activity during Sporulation of Bacillus subtilis

2004 ◽  
Vol 186 (7) ◽  
pp. 1983-1990 ◽  
Author(s):  
David W. Hilbert ◽  
Vasant K. Chary ◽  
Patrick J. Piggot
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Cell Fates ◽  
Gene Encoding ◽  
Primitive Form ◽  
Small Proteins ◽  
Dna Translocase

ABSTRACT Spore formation by Bacillus subtilis is a primitive form of development. In response to nutrient starvation and high cell density, B. subtilis divides asymmetrically, resulting in two cells with different sizes and cell fates. Immediately after division, the transcription factor σF becomes active in the smaller prespore, which is followed by the activation of σE in the larger mother cell. In this report, we examine the role of the mother cell-specific transcription factor σE in maintaining the compartmentalization of gene expression during development. We have studied a strain with a deletion of the spoIIIE gene, encoding a DNA translocase, that exhibits uncompartmentalized σF activity. We have determined that the deletion of spoIIIE alone does not substantially impact compartmentalization, but in the spoIIIE mutant, the expression of putative peptidoglycan hydrolases under the control of σE in the mother cell destroys the integrity of the septum. As a consequence, small proteins can cross the septum, thereby abolishing compartmentalization. In addition, we have found that in a mutant with partially impaired control of σF, the activation of σE in the mother cell is important to prevent the activation of σF in this compartment. Therefore, the activity of σE can either maintain or abolish the compartmentalization of σF, depending upon the genetic makeup of the strain. We conclude that σE activity must be carefully regulated in order to maintain compartmentalization of gene expression during development.

scholarly journals Comparative Transcriptomics Highlights the Role of the Activator Protein 1 Transcription Factor in the Host Response to Ebolavirus

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
James W. Wynne ◽  
Shawn Todd ◽  
Victoria Boyd ◽  
Mary Tachedjian ◽  
Reuben Klein ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Human Cells ◽  
Activator Protein 1 ◽  
Host Gene Expression ◽  
Rna Seq ◽  
Content Type ◽  
Molecular Events ◽  
Ebov Infection

ABSTRACT Ebolavirus and Marburgvirus comprise two genera of negative-sense single-stranded RNA viruses that cause severe hemorrhagic fevers in humans. Despite considerable research efforts, the molecular events following Ebola virus (EBOV) infection are poorly understood. With the view of identifying host factors that underpin EBOV pathogenesis, we compared the transcriptomes of EBOV-infected human, pig, and bat kidney cells using a transcriptome sequencing (RNA-seq) approach. Despite a significant difference in viral transcription/replication between the cell lines, all cells responded to EBOV infection through a robust induction of extracellular growth factors. Furthermore, a significant upregulation of activator protein 1 (AP1) transcription factor complex members FOS and JUN was observed in permissive cell lines. Functional studies focusing on human cells showed that EBOV infection induces protein expression, phosphorylation, and nuclear accumulation of JUN and, to a lesser degree, FOS. Using a luciferase-based reporter, we show that EBOV infection induces AP1 transactivation activity within human cells at 48 and 72 h postinfection. Finally, we show that JUN knockdown decreases the expression of EBOV-induced host gene expression. Taken together, our study highlights the role of AP1 in promoting the host gene expression profile that defines EBOV pathogenesis. IMPORTANCE Many questions remain about the molecular events that underpin filovirus pathophysiology. The rational design of new intervention strategies, such as postexposure therapeutics, will be significantly enhanced through an in-depth understanding of these molecular events. We believe that new insights into the molecular pathogenesis of EBOV may be possible by examining the transcriptomic response of taxonomically diverse cell lines (derived from human, pig, and bat). We first identified the responsive pathways using an RNA-seq-based transcriptomics approach. Further functional and computational analysis focusing on human cells highlighted an important role for the AP1 transcription factor in mediating the transcriptional response to EBOV infection. Our study sheds new light on how host transcription factors respond to and promote the transcriptional landscape that follows viral infection.

scholarly journals The β-Arrestin-Like Protein Rim8 Is Hyperphosphorylated and Complexes with Rim21 and Rim101 To Promote Adaptation to Neutral-Alkaline pH

2012 ◽  
Vol 11 (5) ◽  
pp. 683-693 ◽  
Author(s):  
Jonathan Gomez-Raja ◽  
Dana A. Davis
Keyword(s):  
Signal Transduction ◽  
Transcription Factor ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Multivesicular Bodies ◽  
Ph Sensing ◽  
Content Type ◽  
Protein Levels ◽  
G Protein Coupled

ABSTRACTβ-Arrestin proteins are critical for G-protein-coupled receptor desensitization and turnover. However, β-arrestins have recently been shown to play direct roles in nonheterotrimeric G-protein signal transduction. TheCandida albicansβ-arrestin-like protein Rim8 is required for activation of the Rim101 pH-sensing pathway and for pathogenesis. We have found thatC. albicansRim8 is posttranslationally modified by phosphorylation and specific phosphorylation states are associated with activation of the pH-sensing pathway. Rim8 associated with both the receptor Rim21 and the transcription factor Rim101, suggesting that Rim8 bridges the signaling and activation steps of the pathway. Finally, upon activation of the Rim101 transcription factor,C. albicansRim8 was transcriptionally repressed and Rim8 protein levels were rapidly reduced. Our studies suggest that Rim8 is taken up into multivesicular bodies and degraded within the vacuole. In total, our results reveal a novel mechanism for tightly regulating the activity of a signal transduction pathway. Although the role of β-arrestin proteins in mammalian signal transduction pathways has been demonstrated, relatively little is known about how β-arrestins contribute to signal transduction. Our analyses provide some insights into potential roles.

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