scholarly journals The Integrity of the Cell Wall and Its Remodeling during Heterocyst Differentiation Are Regulated by Phylogenetically Conserved Small RNA Yfr1 in Nostoc sp. Strain PCC 7120

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Manuel Brenes-Álvarez ◽  
Agustín Vioque ◽  
Alicia M. Muro-Pastor
Keyword(s):  
Cell Wall ◽  
Outer Membrane ◽  
Small Rna ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Site Directed Mutagenesis ◽  
Heterocyst Differentiation ◽  
Content Type ◽  
Bacterial Physiology ◽  
Pcc 7120

ABSTRACT Yfr1 is a strictly conserved small RNA in cyanobacteria. A bioinformatic prediction to identify possible interactions of Yfr1 with mRNAs was carried out by using the sequences of Yfr1 from several heterocyst-forming strains, including Nostoc sp. strain PCC 7120. The results of the prediction were enriched in genes encoding outer membrane proteins and enzymes related to peptidoglycan biosynthesis and turnover. Heterologous expression assays with Escherichia coli demonstrated direct interactions of Yfr1 with mRNAs of 11 of the candidate genes. The expression of 10 of them (alr2458, alr4550, murC, all4829, all2158, mraY, alr2269, alr0834, conR, patN) was repressed by interaction with Yfr1, whereas the expression of amiC2, encoding an amidase, was increased. The interactions between Yfr1 and the 11 mRNAs were confirmed by site-directed mutagenesis of Yfr1. Furthermore, a Nostoc strain with reduced levels of Yfr1 had larger amounts of mraY and murC mRNAs, supporting a role for Yfr1 in the regulation of those genes. Nostoc strains with either reduced or increased expression of Yfr1 showed anomalies in cell wall completion and were more sensitive to vancomycin than the wild-type strain. Furthermore, growth in the absence of combined nitrogen, which involves the differentiation of heterocysts, was compromised in the strain overexpressing Yfr1, and filaments were broken at the connections between vegetative cells and heterocysts. These results indicate that Yfr1 is an important regulator of cell wall homeostasis and correct cell wall remodeling during heterocyst differentiation. IMPORTANCE Bacterial small RNAs (sRNAs) are important players affecting the regulation of essentially every aspect of bacterial physiology. The cell wall is a highly dynamic structure that protects bacteria from their fluctuating environment. Cell envelope remodeling is particularly critical for bacteria that undergo differentiation processes, such as spore formation or differentiation of heterocysts. Heterocyst development involves the deposition of additional layers of glycolipids and polysaccharides outside the outer membrane. Here, we show that a cyanobacterial phylogenetically conserved small regulatory RNA, Yfr1, coordinates the expression of proteins involved in cell wall-related processes, including peptidoglycan metabolism and transport of different molecules, as well as expression of several proteins involved in heterocyst differentiation.

scholarly journals Small RNA Regulation of TolC, the Outer Membrane Component of Bacterial Multidrug Transporters

2016 ◽  
Vol 198 (7) ◽  
pp. 1101-1113 ◽  
Author(s):  
Ashley Parker ◽  
Susan Gottesman
Keyword(s):  
Stationary Phase ◽  
Outer Membrane ◽  
Small Rna ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Efflux Pumps ◽  
Toxic Compounds ◽  
Content Type ◽  
Phenotypic Microarray ◽  
Mrna Targets

ABSTRACTBacteria use multidrug efflux pumps to export drugs and toxic compounds out of the cell. One of the most important efflux pumps inEscherichia coliis the AcrAB-TolC system. Small regulatory RNAs (sRNAs) are known to be major posttranscriptional regulators that can enhance or repress translation by binding to the 5′ untranslated region (UTR) of mRNA targets with the help of a chaperone protein, Hfq. In this study, we investigated the expression ofacrA,acrB, andtolCtranslational fusions using 27 Hfq-dependent sRNAs overexpressed from plasmids. No significant sRNA regulation ofacrAoracrBwas detected. SdsR (also known as RyeB), an abundant and well-conserved stationary-phase sRNA, was found to repress the expression oftolC, the gene encoding the outer membrane protein of many multidrug resistance efflux pumps. This repression was shown to be by direct base pairing occurring upstream from the ribosomal binding site. SdsR overexpression and its regulation oftolCwere found to reduce resistance to novobiocin and crystal violet. Our results suggest that additional targets for SdsR exist that contribute to increased antibiotic sensitivity and reduced biofilm formation. In an effort to identify phenotypes associated with single-copy SdsR and its regulation oftolC, the effect of a deletion ofsdsRor mutations intolCthat should block SdsR pairing were investigated using a Biolog phenotypic microarray. However, no significant phenotypes were identified. Therefore, SdsR appears to modulate rather than act as a major regulator of its targets.IMPORTANCEAcrAB-TolC is a major efflux pump present inE. coliand Gram-negative bacteria used to export toxic compounds; the pump confers resistance to many antibiotics of unrelated classes. In this study, we found that SdsR, a small RNA expressed in stationary phase, repressed the expression oftolC, resulting in increased sensitivity to some antibiotics. This extends the findings of previous studies showing that sRNAs contribute to the regulation of many outer membrane proteins; manipulating or enhancing their action might help in sensitizing bacteria to antibiotics.

scholarly journals Establishment of a Protein Concentration Gradient in the Outer Membrane Requires Two Diffusion-Limiting Mechanisms

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Luis David Ginez ◽  
Aurora Osorio ◽  
Laura Camarena ◽  
Sebastian Poggio
Keyword(s):  
Cell Wall ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Concentration Gradient ◽  
Protein Concentration ◽  
Caulobacter Crescentus ◽  
Outer Membrane Proteins ◽  
Structural Domains ◽  
Content Type ◽  
Terminal Domain

ABSTRACTOmpA-like proteins are involved in the stabilization of the outer membrane, resistance to osmotic stress, and pathogenesis. InCaulobacter crescentus, OmpA2 forms a physiologically relevant concentration gradient that forms by an uncharacterized mechanism, in which the gradient orientation depends on the position of the gene locus. This suggests that OmpA2 is synthesized and translocated to the periplasm close to the position of the gene and that the gradient forms by diffusion of the protein from this point. To further understand how the OmpA2 gradient is established, we determined the localization and mobility of the full protein and of its two structural domains. We show that OmpA2 does not diffuse and that both domains are required for gradient formation. The C-terminal domain binds tightly to the cell wall and the immobility of the full protein depends on the binding of this domain to the peptidoglycan; in contrast, the N-terminal membrane β-barrel diffuses slowly. Our results support a model in which once OmpA2 is translocated to the periplasm, the N-terminal membrane β-barrel is required for an initial fast restriction of diffusion until the position of the protein is stabilized by the binding of the C-terminal domain to the cell wall. The implications of these results on outer membrane protein diffusion and organization are discussed.IMPORTANCEProtein concentration gradients play a relevant role in the organization of the bacterial cell. TheCaulobacter crescentusprotein OmpA2 forms an outer membrane polar concentration gradient. To understand the molecular mechanism that determines the formation of this gradient, we characterized the mobility and localization of the full protein and of its two structural domains an integral outer membrane β-barrel and a periplasmic peptidoglycan binding domain. Each domain has a different role in the formation of the OmpA2 gradient, which occurs in two steps. We also show that the OmpA2 outer membrane β-barrel can diffuse, which is in contrast to what has been reported previously for several integral outer membrane proteins inEscherichia coli, suggesting a different organization of the outer membrane proteins.

scholarly journals Novel Antibacterial Targets and Compounds Revealed by a High-Throughput Cell Wall Reporter Assay

2015 ◽  
Vol 197 (10) ◽  
pp. 1726-1734 ◽  
Author(s):  
Asha S. Nayar ◽  
Thomas J. Dougherty ◽  
Keith E. Ferguson ◽  
Brett A. Granger ◽  
Lisa McWilliams ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Outer Membrane ◽  
High Throughput ◽  
Cell Envelope ◽  
Mechanisms Of Action ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Phenotypic Screen

ABSTRACTA high-throughput phenotypic screen based on aCitrobacter freundiiAmpC reporter expressed inEscherichia coliwas executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action.E. colimutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments withE. colispheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy.IMPORTANCEThe prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targetingEscherichia coliouter membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.

scholarly journals c-di-AMP Accumulation Impairs Muropeptide Synthesis in Listeria monocytogenes

2020 ◽  
Vol 202 (24) ◽  
Author(s):  
Steven M. Massa ◽  
Amar Deep Sharma ◽  
Cheta Siletti ◽  
Zepeng Tu ◽  
Jared J. Godfrey ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Envelope ◽  
Second Messenger ◽  
Fold Increase ◽  
Bacterial Cells ◽  
Content Type ◽  
Bacterial Physiology ◽  
Peptidoglycan Synthesis ◽  
Increased Sensitivity

ABSTRACT Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger among bacteria. c-di-AMP regulates many cellular pathways through direct binding to several molecular targets in bacterial cells. c-di-AMP depletion is well known to destabilize the bacterial cell wall, resulting in increased bacteriolysis and enhanced susceptibility to cell wall targeting antibiotics. Using the human pathogen Listeria monocytogenes as a model, we found that c-di-AMP accumulation also impaired cell envelope integrity. An L. monocytogenes mutant deleted for c-di-AMP phosphodiesterases (pdeA pgpH mutant) exhibited a 4-fold increase in c-di-AMP levels and several cell wall defects. For instance, the pdeA pgpH mutant was defective for the synthesis of peptidoglycan muropeptides and was susceptible to cell wall-targeting antimicrobials. Among different muropeptide precursors, we found that the pdeA pgpH strain was particularly impaired in the synthesis of d-Ala–d-Ala, which is required to complete the pentapeptide stem associated with UDP–N-acetylmuramic acid (MurNAc). This was consistent with an increased sensitivity to d-cycloserine, which inhibits the d-alanine branch of peptidoglycan synthesis. Finally, upon examining d-Ala:d-Ala ligase (Ddl), which catalyzes the conversion of d-Ala to d-Ala–d-Ala, we found that its activity was activated by K+. Based on previous reports that c-di-AMP inhibits K+ uptake, we propose that c-di-AMP accumulation impairs peptidoglycan synthesis, partially through the deprivation of cytoplasmic K+ levels, which are required for cell wall-synthetic enzymes. IMPORTANCE The bacterial second messenger c-di-AMP is produced by a large number of bacteria and conditionally essential to many species. Conversely, c-di-AMP accumulation is also toxic to bacterial physiology and pathogenesis, but its mechanisms are largely undefined. We found that in Listeria monocytogenes, elevated c-di-AMP levels diminished muropeptide synthesis and increased susceptibility to cell wall-targeting antimicrobials. Cell wall defects might be an important mechanism for attenuated virulence in bacteria with high c-di-AMP levels.

scholarly journals Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Peter Mellroth ◽  
Tatyana Sandalova ◽  
Alexey Kikhney ◽  
Francisco Vilaplana ◽  
Dusan Hesek ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Solution Structure ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Lytic Activity ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Content Type ◽  
Peptidoglycan Synthesis

ABSTRACT The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

scholarly journals NsiR1 , a small RNA with multiple copies, modulates heterocyst differentiation in the cyanobacterium Nostoc sp. PCC 7120

2020 ◽  
Vol 22 (8) ◽  
pp. 3325-3338 ◽  
Author(s):  
Manuel Brenes‐Álvarez ◽  
Marina Minguet ◽  
Agustín Vioque ◽  
Alicia M. Muro‐Pastor
Keyword(s):  
Small Rna ◽  
Heterocyst Differentiation ◽  
Multiple Copies ◽  
Pcc 7120

scholarly journals The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance inClostridium difficile

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Elodie Cuenot ◽  
Transito Garcia-Garcia ◽  
Thibaut Douche ◽  
Olivier Gorgette ◽  
Pascal Courtin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
Hamster Model ◽  
Content Type ◽  
Physiological Processes ◽  
Signal Transduction Networks ◽  
Mean Size

ABSTRACTClostridium difficileis the leading cause of antibiotic-associated diarrhea in adults. During infection,C. difficilemust detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC ofC. difficileis an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion ofprkCaffects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkCmutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkCmutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkCmutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority ofC. difficileproteins associated with the cell wall were less abundant in the ΔprkCmutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkCmutant had a colonization delay that did not significantly affect overall virulence.

scholarly journals A Disulfide Oxidoreductase (CHU_1165) Is Essential for Cellulose Degradation by Affecting Outer Membrane Proteins in Cytophaga hutchinsonii

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Dong Zhao ◽  
Ying Wang ◽  
Sen Wang ◽  
Weican Zhang ◽  
Qingsheng Qi ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Disulfide Bond ◽  
Cellulose Degradation ◽  
Outer Membrane Proteins ◽  
Hypothetical Protein ◽  
Pleiotropic Effect ◽  
Content Type ◽  
Cytophaga Hutchinsonii ◽  
Disulfide Oxidoreductase

ABSTRACT Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii. Further study showed that cells of the Δ1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro. Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins. IMPORTANCE Cytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii. Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

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