scholarly journals Coxiella burnetiiRpoS Regulates Genes Involved in Morphological Differentiation and Intracellular Growth

2019 ◽  
Vol 201 (8) ◽  
Author(s):  
Derek E. Moormeier ◽  
Kelsi M. Sandoz ◽  
Paul A. Beare ◽  
Daniel E. Sturdevant ◽  
Vinod Nair ◽  
...  
Keyword(s):  
Cell Wall ◽  
Stationary Phase ◽  
Stress Responses ◽  
Q Fever ◽  
Morphological Differentiation ◽  
Developmental Cycle ◽  
Intracellular Growth ◽  
Content Type ◽  
Small Cell Variant ◽  
Cell Variant

ABSTRACTCoxiella burnetii, the etiological agent of Q fever, undergoes a unique biphasic developmental cycle where bacteria transition from a replicating (exponential-phase) large cell variant (LCV) form to a nonreplicating (stationary-phase) small cell variant (SCV) form. The alternative sigma factor RpoS is an essential regulator of stress responses and stationary-phase physiology in several bacterial species, includingLegionella pneumophila, which has a developmental cycle superficially similar to that ofC. burnetii. Here, we used aC. burnetiiΔrpoSmutant to define the role of RpoS in intracellular growth and SCV development. Growth yields following infection of Vero epithelial cells or THP-1 macrophage-like cells with therpoSmutant in the SCV form, but not the LCV form, were significantly lower than that of wild-type bacteria. RNA sequencing and whole-cell mass spectrometry of theC. burnetiiΔrpoSmutant revealed that a substantial portion of theC. burnetiigenome is regulated by RpoS during SCV development. Regulated genes include those involved in stress responses, arginine transport, peptidoglycan remodeling, and synthesis of the SCV-specific protein ScvA. Genes comprising thedot/icmlocus, responsible for production of the Dot/Icm type 4B secretion system, were also dysregulated in therpoSmutant. These data were corroborated with independent assays demonstrating that theC. burnetiiΔrpoSstrain has increased sensitivity to hydrogen peroxide and carbenicillin and a thinner cell wall/outer membrane complex. Collectively, these results demonstrate that RpoS is an important regulator of genes involved inC. burnetiiSCV development and intracellular growth.IMPORTANCEThe Q fever bacteriumCoxiella burnetiihas spore-like environmental stability, a characteristic that contributes to its designation as a potential bioweapon. Stability is likely conferred by a highly resistant, small cell variant (SCV) stationary-phase form that arises during a biphasic developmental cycle. Here, we define the role of the alternative sigma factor RpoS in regulating genes associated with SCV development. Genes involved in stress responses, amino acid transport, cell wall remodeling, and type 4B effector secretion were dysregulated in therpoSmutant. Cellular impairments included defects in intracellular growth, cell wall structure, and resistance to oxidants. These results support RpoS as a central regulator of theCoxielladevelopmental cycle and identify developmentally regulated genes involved in morphological differentiation.

scholarly journals The Nature of Antibacterial Adaptive Immune Responses against Staphylococcus aureus Is Dependent on the Growth Phase and Extracellular Peptidoglycan

2019 ◽  
Vol 88 (1) ◽  
Author(s):  
Payal P. Balraadjsing ◽  
Lisbeth D. Lund ◽  
Yuri Souwer ◽  
Sebastian A. J. Zaat ◽  
Hanne Frøkiær ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
T Cell ◽  
Stationary Phase ◽  
Growth Phase ◽  
Cell Response ◽  
Th17 Cell ◽  
Exponential Phase ◽  
Th1 Cell ◽  
Content Type

ABSTRACT Staphylococcus aureus has evolved different strategies to evade the immune response, which play an important role in its pathogenesis. The bacteria express and shed various cell wall components and toxins during different stages of growth that may affect the protective T cell responses to extracellular and intracellular S. aureus. However, if and how the dendritic cell (DC)-mediated T cell response against S. aureus changes during growth of the bacterium remain elusive. In this study, we show that exponential-phase (EP) S. aureus bacteria were endocytosed very efficiently by human DCs, and these DCs strongly promoted production of the T cell polarizing factor interleukin-12 (IL-12). In contrast, stationary-phase (SP) S. aureus bacteria were endocytosed less efficiently by DCs, and these DCs produced small amounts of IL-12. The high level of IL-12 production induced by EP S. aureus led to the development of a T helper 1 (Th1) cell response, which was inhibited after neutralization of IL-12. Furthermore, preincubation with the staphylococcal cell wall component peptidoglycan (PGN), characteristically shed during the exponential growth phase, modulated the DC response to EP S. aureus. PGN preincubation appeared to inhibit IL-12p35 expression, leading to downregulation of IL-12 and an increase of IL-23 production by DCs, enhancing Th17 cell development. Taken together, our data indicate that exponential-phase S. aureus bacteria induce a stronger IL-12-dependent Th1 cell response than stationary-phase S. aureus and that this Th1 cell response shifted toward a Th17 cell response in the presence of PGN.

scholarly journals High-Resolution Analysis of the Peptidoglycan Composition inStreptomyces coelicolor

2018 ◽  
Vol 200 (20) ◽  
Author(s):  
Lizah T. van der Aart ◽  
Gerwin K. Spijksma ◽  
Amy Harms ◽  
Waldemar Vollmer ◽  
Thomas Hankemeier ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycelial Growth ◽  
Antibiotic Production ◽  
Hydrolytic Enzymes ◽  
Morphological Differentiation ◽  
Model Organisms ◽  
Major Constituent ◽  
Single Spore ◽  
Content Type ◽  
Liquid Chromatography Tandem Mass

ABSTRACTThe bacterial cell wall maintains cell shape and protects against bursting by turgor. A major constituent of the cell wall is peptidoglycan (PG), which is continuously modified to enable cell growth and differentiation through the concerted activity of biosynthetic and hydrolytic enzymes. Streptomycetes are Gram-positive bacteria with a complex multicellular life style alternating between mycelial growth and the formation of reproductive spores. This involves cell wall remodeling at apical sites of the hyphae during cell elongation and autolytic degradation of the vegetative mycelium during the onset of development and antibiotic production. Here, we show that there are distinct differences in the cross-linking and maturation of the PGs between exponentially growing vegetative hyphae and the aerial hyphae that undergo sporulation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified over 80 different muropeptides, revealing that major PG hydrolysis takes place over the course of mycelial growth. Half of the dimers lacked one of the disaccharide units in transition-phase cells, most likely due to autolytic activity. The deacetylation of MurNAc to MurN was particularly pronounced in spores and strongly reduced in sporulation mutants with a deletion ofbldDorwhiG, suggesting that MurN is developmentally regulated. Altogether, our work highlights the dynamic and growth phase-dependent changes in the composition of the PG inStreptomyces.IMPORTANCEStreptomycetes are bacteria with a complex lifestyle and are model organisms for bacterial multicellularity. From a single spore, a large multigenomic multicellular mycelium is formed, which differentiates to form spores. Programmed cell death is an important event during the onset of morphological differentiation. In this work, we provide new insights into the changes in the peptidoglycan composition and over time, highlighting changes over the course of development and between growing mycelia and spores. This revealed dynamic changes in the peptidoglycan when the mycelia aged, with extensive peptidoglycan hydrolysis and, in particular, an increase in the proportion of 3-3 cross-links. Additionally, we identified a muropeptide that accumulates predominantly in the spores and may provide clues toward spore development.

scholarly journals Carbon Storage Regulator A Contributes to the Virulence of Haemophilus ducreyi in Humans by Multiple Mechanisms

2012 ◽  
Vol 81 (2) ◽  
pp. 608-617 ◽  
Author(s):  
Dharanesh Gangaiah ◽  
Wei Li ◽  
Kate R. Fortney ◽  
Diane M. Janowicz ◽  
Sheila Ellinger ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stationary Phase ◽  
Carbon Storage ◽  
Stress Responses ◽  
Haemophilus Ducreyi ◽  
Content Type ◽  
Enhanced Sensitivity ◽  
Significant Survival ◽  
Carbon Storage Regulator ◽  
Mutant Phenotypes

ABSTRACTThe carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence.Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog ofcsrA. Here, we generated an unmarked, in-frame deletion mutant ofcsrAto assess its contribution toH. ducreyipathogenesis. In human inoculation experiments, thecsrAmutant was partially attenuated for pustule formation compared to its parent. Deletion ofcsrAresulted in decreased adherence ofH. ducreyito human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants ofH. ducreyiadherence to HFF cells, were downregulated in thecsrAmutant. Compared to its parent, thecsrAmutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. ThecsrAmutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation intranspartially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.

scholarly journals Appropriate Regulation of the σE-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Hervé Nicoloff ◽  
Saumya Gopalkrishnan ◽  
Sarah E. Ades
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Outer Membrane ◽  
Stress Responses ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Point Mutations ◽  
Content Type ◽  
Envelope Stress ◽  
Outer Membrane Porins

ABSTRACT The alternative sigma factor σE is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σE increases during entry into stationary phase, suggesting an important role for σE when nutrients are limiting. Elevated σE activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand σE-directed cell lysis and the role of σE in stationary phase, we investigated the effects of elevated σE activity in cultures grown for 10 days. We demonstrate that high σE activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced σE activity, due primarily to point mutations in the region of σE that binds the −35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High σE activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg2+ and buffering the growth medium or by deleting the σE-dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of σE activity. Our results demonstrate that appropriate regulation of σE activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability. IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The σE response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of σE is tightly regulated to match the production of σE regulon members to the needs of the cell. In E. coli, loss of σE results in lethality. Here we demonstrate that excessive σE activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent.

scholarly journals Complex Physiology and Compound Stress Responses during Fermentation of Alkali-Pretreated Corn Stover Hydrolysate by an Escherichia coli Ethanologen

2012 ◽  
Vol 78 (9) ◽  
pp. 3442-3457 ◽  
Author(s):  
Michael S. Schwalbach ◽  
David H. Keating ◽  
Mary Tremaine ◽  
Wesley D. Marner ◽  
Yaoping Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acids ◽  
Stationary Phase ◽  
Gene Expression Profiling ◽  
Stress Responses ◽  
Expression Profiling ◽  
Content Type ◽  
E Coli ◽  
Cell Maintenance

ABSTRACTThe physiology of ethanologenicEscherichia coligrown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into howE. coliresponds to such hydrolysates, we studied anE. coliK-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate,E. coliceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.

scholarly journals Polydiglycosylphosphate Transferase PdtA (SCO2578) of Streptomyces coelicolor A3(2) Is Crucial for Proper Sporulation and Apical Tip Extension under Stress Conditions

2016 ◽  
Vol 82 (18) ◽  
pp. 5661-5672 ◽  
Author(s):  
Steffen Sigle ◽  
Nadja Steblau ◽  
Wolfgang Wohlleben ◽  
Günther Muth
Keyword(s):  
Cell Wall ◽  
Life Cycle ◽  
Vegetative Growth ◽  
Cell Envelope ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Spore Wall ◽  
Stress Conditions ◽  
Content Type ◽  
Tip Extension

ABSTRACTAlthough anionic glycopolymers are crucial components of the Gram-positive cell envelope, the relevance of anionic glycopolymers for vegetative growth and morphological differentiation ofStreptomyces coelicolorA3(2) is unknown. Here, we show that the LytR-CpsA-Psr (LCP) protein PdtA (SCO2578), a TagV-like glycopolymer transferase, has a dual function in theS. coelicolorA3(2) life cycle. Despite the presence of 10 additional LCP homologs, PdtA is crucial for proper sporulation. The integrity of the spore envelope was severely affected in apdtAdeletion mutant, resulting in 34% nonviable spores.pdtAdeletion caused a significant reduction in the polydiglycosylphosphate content of the spore envelope. Beyond that, apical tip extension and normal branching of vegetative mycelium were severely impaired on high-salt medium. This growth defect coincided with the mislocalization of peptidoglycan synthesis. Thus, PdtA itself or the polydiglycosylphosphate attached to the peptidoglycan by the glycopolymer transferase PdtA also has a crucial function in apical tip extension of vegetative hyphae under stress conditions.IMPORTANCEAnionic glycopolymers are underappreciated components of the Gram-positive cell envelope. They provide rigidity to the cell wall and position extracellular enzymes involved in peptidoglycan remodeling. AlthoughStreptomyces coelicolorA3(2), the model organism for bacterial antibiotic production, is known to produce two distinct cell wall-linked glycopolymers, teichulosonic acid and polydiglycosylphosphate, the role of these glycopolymers in theS. coelicolorA3(2) life cycle has not been addressed so far. This study reveals a crucial function of the anionic glycopolymer polydiglycosylphosphate for the growth and morphological differentiation ofS. coelicolorA3(2). Polydiglycosylphosphate is attached to the spore wall by the LytR-CpsA-Psr protein PdtA (SCO2578), a component of theStreptomycesspore wall-synthesizing complex (SSSC), to ensure the integrity of the spore envelope. Surprisingly, PdtA also has a crucial role in vegetative growth under stress conditions and is required for proper peptidoglycan incorporation during apical tip extension.

scholarly journals Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12

2017 ◽  
Vol 199 (7) ◽  
Author(s):  
Garrett T. Wong ◽  
Richard P. Bonocora ◽  
Alicia N. Schep ◽  
Suzannah M. Beeler ◽  
Anna J. Lee Fong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Stress Responses ◽  
Sigma Factor ◽  
Core Promoter ◽  
Transcriptional Responses ◽  
Content Type ◽  
Cellular Processes ◽  
K 12 ◽  
Sigma Factor Rpos

ABSTRACT The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli. The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS (“sensitive” genes), and genes whose expression changes very little with the production of a little RpoS (“insensitive”). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS. IMPORTANCE The sigma factor RpoS is a global regulator that controls the response to many stresses in Escherichia coli. Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism that coordinates specific cellular processes in response to stresses.

scholarly journals Coxiella burnetii Exhibits Morphological Change and Delays Phagolysosomal Fusion after Internalization by J774A.1 Cells

2000 ◽  
Vol 68 (7) ◽  
pp. 3815-3821 ◽  
Author(s):  
Dale Howe ◽  
Louis P. Mallavia
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Large Cell ◽  
Infection Process ◽  
Thorium Dioxide ◽  
Small Cell Variant ◽  
Morphological Variants ◽  
Cell Variant ◽  
First Time

ABSTRACT Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium proliferating within the harsh environment of the phagolysosome. Mechanisms controlling trafficking to, and survival of pathogens within, the phagolysosome are unknown. Two distinct morphological variants have been implicated as playing a role in C. burnetii survival. The dormant small-cell variant (SCV) is resistant to extracellular stresses and the more metabolically active large-cell variant (LCV) is sensitive to environmental stresses. To document changes in the ratio of SCVs to LCVs in response to environment, a protein specific to SCV, ScvA, was quantitated. During the first 2 h after internalization ofC. burnetii by J774A.1 cells, the level of ScvA decreased, indicating a change from a population containing primarily SCVs to one containing primarily LCVs. In vitro experiments showed that 2 h of incubation at pH 5.5 caused a significant decrease in ScvA in contrast to incubation at pH 4.5. Measuring in vitro internalization of [35S]methionine-[35S]cysteine in response to pH, we found the uptake to be optimal at pH 5.5. To explore the possibility that after uptake C. burnetii was able to delay phagolysosomal fusion, we used thorium dioxide and acid phosphatase to label phagolysosomes during infection of J774A.1 cells. We determined that viable C. burnetii was able to delay phagolysosomal fusion. This is the first time that a delay in phagolysosomal fusion has been shown to be a part of the infection process of this pathogenic microorganism.

scholarly journals Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form

PLoS ONE ◽  
2016 ◽  
Vol 11 (2) ◽  
pp. e0149957 ◽  
Author(s):  
Kelsi M. Sandoz ◽  
David L. Popham ◽  
Paul A. Beare ◽  
Daniel E. Sturdevant ◽  
Bryan Hansen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Coxiella Burnetii ◽  
Transcriptional Profiling ◽  
Small Cell ◽  
Small Cell Variant ◽  
Cell Wall Remodeling ◽  
Extensive Cell ◽  
Cell Variant ◽  
Developmental Form

scholarly journals Contribution of Stress Responses to Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms

2015 ◽  
Vol 59 (7) ◽  
pp. 3838-3847 ◽  
Author(s):  
Philip S. Stewart ◽  
Michael J. Franklin ◽  
Kerry S. Williamson ◽  
James P. Folsom ◽  
Laura Boegli ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stationary Phase ◽  
Stress Responses ◽  
Cell Physiology ◽  
Wild Type ◽  
Phase Growth ◽  
Biofilm Growth ◽  
Content Type ◽  
Hypoxia Stress ◽  
Biofilm Bacteria

ABSTRACTEnhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth ofPseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient inrpoS,relAandspoT, oranrwere cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of aP. aeruginosabiofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin.

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