scholarly journals Role of LmeA, a Mycobacterial Periplasmic Protein, in Maintaining the Mannosyltransferase MptA and Its Product Lipomannan under Stress

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Kathryn C. Rahlwes ◽  
Sarah H. Osman ◽  
Yasu S. Morita
Keyword(s):  
Growth Phase ◽  
Molecular Mechanisms ◽  
Cell Envelope ◽  
Stationary Growth Phase ◽  
Cellular Level ◽  
Nutrient Starvation ◽  
Stress Conditions ◽  
Periplasmic Protein ◽  
Stationary Growth

ABSTRACT The mycobacterial cell envelope has a diderm structure, composed of an outer mycomembrane, an arabinogalactan-peptidoglycan cell wall, a periplasm, and an inner membrane. Lipomannan (LM) and lipoarabinomannan (LAM) are structural and immunomodulatory components of this cell envelope. LM/LAM biosynthesis involves a number of mannosyltransferases and acyltransferases, and MptA is an α1,6-mannosyltransferase involved in the final extension of the mannan chain. Recently, we reported the periplasmic protein LmeA being involved in the maturation of the mannan backbone in Mycobacterium smegmatis. Here, we examined the role of LmeA under stress conditions. We found that lmeA transcription was upregulated under two stress conditions: stationary growth phase and nutrient starvation. Under both conditions, LAM was decreased, but LM was relatively stable, suggesting that maintaining the cellular level of LM under stress is important. Surprisingly, the protein levels of MptA were decreased in an lmeA deletion (ΔlmeA) mutant under both stress conditions. The transcript levels of mptA in the ΔlmeA mutant were similar to or even higher than those in the wild type, indicating that the decrease of MptA protein was a posttranscriptional event. The ΔlmeA mutant was unable to maintain the cellular level of LM under stress, consistent with the decrease in MptA. Even during active growth, overexpression of LmeA led the cells to produce more LM and become more resistant to several antibiotics. Altogether, our study reveals the roles of LmeA in the homeostasis of the MptA mannosyltransferase, particularly under stress conditions, ensuring the stable expression of LM and the maintenance of cell envelope integrity. IMPORTANCE Mycobacteria differentially regulate the cellular amounts of lipoglycans in response to environmental changes, but the molecular mechanisms of this regulation remain unknown. Here, we demonstrate that cellular lipoarabinomannan (LAM) levels rapidly decline under two stress conditions, stationary growth phase and nutrient starvation, while the levels of another related lipoglycan, lipomannan (LM), stay relatively constant. The persistence of LM under stress correlated with the maintenance of two key mannosyltransferases, MptA and MptC, in the LM biosynthetic pathway. We further showed that the stress exposures lead to the upregulation of lmeA gene expression and that the periplasmic protein LmeA plays a key role in maintaining the enzyme MptA and its product LM under stress conditions. These findings reveal new aspects of how lipoglycan biosynthesis is regulated under stress conditions in mycobacteria.

scholarly journals The role of LmeA, a mycobacterial periplasmic protein, in stabilizing the mannosyltransferase MptA and its product lipomannan under stress

2020 ◽  
Author(s):  
Kathryn C. Rahlwes ◽  
Sarah H. Osman ◽  
Yasu S. Morita
Keyword(s):  
Mycobacterium Smegmatis ◽  
Cell Envelope ◽  
Stationary Growth Phase ◽  
Cellular Level ◽  
Stress Conditions ◽  
Periplasmic Protein ◽  
Active Growth ◽  
Protein Levels ◽  
Peptidoglycan Cell Wall

ABSTRACTThe mycobacterial cell envelope has a diderm structure, composed of an outer mycomembrane, an arabinogalactan-peptidoglycan cell wall, periplasm and an inner membrane. Lipomannan (LM) and lipoarabinomannan (LAM) are structural and immunomodulatory components of this cell envelope. LM/LAM biosynthesis involves a number of mannosyltransferases and acyltransferases, and MptA is an α1,6-mannosyltransferase involved in the final extension of the mannan backbones. Recently, we reported the periplasmic protein LmeA being involved in the maturation of the mannan backbone in Mycobacterium smegmatis. Here, we examined the role of LmeA under stress conditions. We found that the lmeA transcription was upregulated under two stress conditions: stationary growth phase and nutrient starvation. Under both conditions, LAM was decreased, but LM was relatively stable, suggesting that maintaining the cellular level of LM under stress is important. Surprisingly, the protein levels of MptA were decreased in lmeA deletion mutant (ΔlmeA) in both stress conditions. The transcript levels of mptA in ΔlmeA were similar to or even higher than those in the wildtype, indicating that the decrease of MptA protein was a post-transcriptional event. Consistent with the decrease in MptA, ΔlmeA was unable to maintain the cellular level of LM under stress. Even during active growth, overexpression of LmeA led the cells to produce more LM and become more resistant to several antibiotics. Altogether, our study reveals the roles of LmeA in the homeostasis of the MptA mannosyltransferase particularly under stress conditions, ensuring the stable expression of LM and the maintenance of cell envelope integrity.

scholarly journals Stress-Induced Reorganization of the Mycobacterial Membrane Domain

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Jennifer M. Hayashi ◽  
Kirill Richardson ◽  
Emily S. Melzer ◽  
Steven J. Sandler ◽  
Bree B. Aldridge ◽  
...  
Keyword(s):  
Growth Phase ◽  
Cell Elongation ◽  
Cell Envelope ◽  
Stationary Growth Phase ◽  
Nutrient Starvation ◽  
Membrane Domain ◽  
Stationary Growth ◽  
Important Species ◽  
Peptidoglycan Biosynthesis ◽  
Mycobacterial Cell

ABSTRACTCell elongation occurs primarily at the mycobacterial cell poles, but the molecular mechanisms governing this spatial regulation remain elusive. We recently reported the presence of an intracellular membrane domain (IMD) that was spatially segregated from the conventional plasma membrane inMycobacterium smegmatis. The IMD is enriched in the polar region of actively elongating cells and houses many essential enzymes involved in envelope biosynthesis, suggesting its role in spatially restricted elongation at the cell poles. Here, we examined reorganization of the IMD when the cells are no longer elongating. To monitor the IMD, we used a previously established reporter strain expressing fluorescent IMD markers and grew it to the stationary growth phase or exposed the cells to nutrient starvation. In both cases, the IMD was delocalized from the cell pole and distributed along the sidewall. Importantly, the IMD could still be isolated biochemically by density gradient fractionation, indicating its maintenance as a membrane domain. Chemical and genetic inhibition of peptidoglycan biosynthesis led to the delocalization of the IMD, suggesting the suppression of peptidoglycan biosynthesis as a trigger of spatial IMD rearrangement. Starved cells with a delocalized IMD can resume growth upon nutrient repletion, and polar enrichment of the IMD coincides with the initiation of cell elongation. These data reveal that the IMD is a membrane domain with the unprecedented capability of subcellular repositioning in response to the physiological conditions of the mycobacterial cell.IMPORTANCEMycobacteria include medically important species, such as the human tuberculosis pathogenMycobacterium tuberculosis. The highly impermeable cell envelope is a hallmark of these microbes, and its biosynthesis is a proven chemotherapeutic target. Despite the accumulating knowledge regarding the biosynthesis of individual envelope components, the regulatory mechanisms behind the coordinated synthesis of the complex cell envelope remain elusive. We previously reported the presence of a metabolically active membrane domain enriched in the elongating poles of actively growing mycobacteria. However, the spatiotemporal dynamics of the membrane domain in response to stress have not been examined. Here, we show that the membrane domain is spatially reorganized when growth is inhibited in the stationary growth phase, under nutrient starvation, or in response to perturbation of peptidoglycan biosynthesis. Our results suggest that mycobacteria have a mechanism to spatiotemporally coordinate the membrane domain in response to metabolic needs under different growth conditions.

scholarly journals Clostridioides difficile Phosphoproteomics Shows an Expansion of Phosphorylated Proteins in Stationary Growth Phase

mSphere ◽  
2022 ◽  
Author(s):  
Wiep Klaas Smits ◽  
Yassene Mohammed ◽  
Arnoud H. de Ru ◽  
Valentina Cordo' ◽  
Annemieke H. Friggen ◽  
...  
Keyword(s):  
Growth Phase ◽  
Posttranslational Modification ◽  
Stationary Growth Phase ◽  
Comprehensive Analysis ◽  
Limited Evidence ◽  
Stationary Growth ◽  
Content Type ◽  
Phosphorylated Proteins ◽  
Clostridioides Difficile

In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile . To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile .  

scholarly journals Role of the Stationary Growth Phase Sigma Factor RpoS of Burkholderia pseudomallei in Response to Physiological Stress Conditions

2003 ◽  
Vol 185 (23) ◽  
pp. 7008-7014 ◽  
Author(s):  
Benchamas Subsin ◽  
Mark S. Thomas ◽  
Gerd Katzenmeier ◽  
Jonathan G. Shaw ◽  
Sumalee Tungpradabkul ◽  
...  
Keyword(s):  
Growth Phase ◽  
Sigma Factor ◽  
Burkholderia Pseudomallei ◽  
Physiological Stress ◽  
Stationary Growth Phase ◽  
Null Mutant ◽  
Stationary Growth ◽  
Increased Sensitivity ◽  
And Oxidative Stress

ABSTRACT The Burkholderia pseudomallei rpoS gene was identified, and an rpoS null mutant was constructed. The mutant was shown to have an increased sensitivity to carbon starvation and oxidative stress. By using rpoS-lacZ fusions, transcription of rpoS was shown to be growth phase regulated, reaching a peak upon entry into stationary phase.

scholarly journals EttA is likely non-essential in Staphylococcus aureus persistence, fitness or resistance to antibiotics

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Michal Meir ◽  
Anna Rozenblit ◽  
Simona Fliger ◽  
Yuval Geffen ◽  
Daniel Barkan
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Growth Phase ◽  
Cell Formation ◽  
Stationary Growth Phase ◽  
Clinical Isolates ◽  
Beta Lactam ◽  
Stationary Growth ◽  
Mechanisms Of Persistence

Abstract Background Tolerance to antibiotics and persistence are associated with antibiotic treatment failures, chronic-relapsing infections, and emerging antibiotic resistance in various bacteria, including Staphylococcus aureus. Mechanisms of persistence are largely unknown, yet have been linked to physiology under low-ATP conditions and the metabolic-inactive state. EttA is an ATP-binding cassette protein, linked in Eschrechia coli to ribosomal hibernation and fitness in stationary growth phase, yet its role in S. aureus physiology is unknown. Results Using whole genome sequencing (WGS) of serial clinical isolates, we identified an EttA-negative S. aureus mutant (ettAstop), and its isogenic wild-type counterpart. We used these two isogenic clones to investigate the role of ettA in S. aureus physiology in starvation and antibiotic stress, and test its role in persistence and antibiotic tolerance. ettAstop and its WT counterpart were similar in their antibiotic resistance profiles to multiple antibiotics. Population dynamics of ettAstop and the WT were similar in low-nutrient setting, with similar recovery from stationary growth phase or starvation. Supra-bacteriocidal concentration of cefazolin had the same killing effect on ettAstop and WT populations, with no difference in persister formation. Conclusions Lack of ettA does not affect S. aureus antibiotic resistance, beta-lactam tolerance, resilience to starvation or fitness following starvation. We conclude the role of ettA in S. aureus physiology is limited or redundant with another, unidentified gene. WGS of serial clinical isolates may enable investigation of other single genes involved in S. aureus virulence, and specifically persister cell formation.

scholarly journals Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine

2008 ◽  
Vol 74 (15) ◽  
pp. 4847-4852 ◽  
Author(s):  
Anastasia Matthies ◽  
Thomas Clavel ◽  
Michael Gütschow ◽  
Wolfram Engst ◽  
Dirk Haller ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Enzyme Induction ◽  
Growth Phase ◽  
Anaerobic Bacterium ◽  
Stationary Growth Phase ◽  
Gut Bacteria ◽  
Soy Isoflavones ◽  
Strictly Anaerobic ◽  
Stationary Growth ◽  
Mouse Intestine

ABSTRACT The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.

scholarly journals The extracellular proteome of Rhizobium etli CE3 in exponential and stationary growth phase

2010 ◽  
Vol 8 (1) ◽  
pp. 51 ◽  
Author(s):  
Niurka Meneses ◽  
Guillermo Mendoza-Hernández ◽  
Sergio Encarnación
Keyword(s):  
Growth Phase ◽  
Stationary Growth Phase ◽  
Rhizobium Etli ◽  
Stationary Growth ◽  
Extracellular Proteome

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 Proteomic Study of the Exponential-Stationary Growth Phase Transition in the Haloarchaea Natrialba magadii and Haloferax volcanii

PROTEOMICS ◽  
2018 ◽  
Vol 18 (14) ◽  
pp. 1800116 ◽  
Author(s):  
Micaela Cerletti ◽  
María Ines Giménez ◽  
Christian Tröetschel ◽  
Celeste D’ Alessandro ◽  
Ansgar Poetsch ◽  
...  
Keyword(s):  
Phase Transition ◽  
Growth Phase ◽  
Stationary Growth Phase ◽  
Haloferax Volcanii ◽  
Stationary Growth ◽  
Proteomic Study ◽  
Natrialba Magadii ◽  
Growth Phase Transition

LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi

2021 ◽  
Author(s):  
yaqiu Zhang ◽  
Yiqing Deng ◽  
Juan Feng ◽  
Jianmei Hu ◽  
Haoxiang Chen ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Stationary Growth Phase ◽  
Extracellular Protease ◽  
Fish Pathogen ◽  
Wild Type ◽  
Stationary Growth ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Luxs Mutant

In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. The statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and the ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and the luxS mutant. However, the deletion of luxS decreased the secretion of extracellular protease, while increased the ability of swimming and swarming. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented the defect. Since motility is flagella dependent, 16 of V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly up-regulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phase.

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