D-alanine substitution of teichoic acids as a modulator of protein folding and stability at the cytoplasmic membrane-cell wall interface of Bacillus subtilis

ABSTRACT The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is d-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for d-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of d-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of d-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.

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Nisin Resistance in Listeria monocytogenes ATCC 700302 Is a Complex Phenotype

Applied and Environmental Microbiology ◽

10.1128/aem.64.1.231-237.1998 ◽

1998 ◽

Vol 64 (1) ◽

pp. 231-237 ◽

Cited By ~ 169

Author(s):

Allison D. Crandall ◽

Thomas J. Montville

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Cytoplasmic Membrane ◽

Divalent Cations ◽

Cross Resistance ◽

Complex Phenotype ◽

In The Wild ◽

Membrane Changes ◽

Lower Ratio ◽

Membrane Cell

ABSTRACT Nisin resistance in Listeria monocytogenes ATCC 700302 is a complex phenotype involving alterations in both the cytoplasmic membrane and the cell wall and a requirement for divalent cations. In addition to a lower ratio of C15 to C17 fatty acids than in the wild-type strain (A. S. Mazzotta and T. J. Montville, J. Appl. Microbiol. 82:32–38, 1997), this nisin-resistant (Nisr) strain contained significantly more zwitterionic phosphatidylethanolamine and less anionic phosphatidylglycerol and cardiolipin. The extraction of cardiolipin was enhanced by a penicillin-lysozyme step to disrupt the cell wall. This study is the first to quantify the phosphatidylethanolamine component of the L. monocytogenes cytoplasmic membrane. While these cytoplasmic membrane changes were induced by nisin, the Nisr strain also showed altered sensitivities to cell wall-acting compounds, even when grown in the absence of nisin, suggesting a constitutive alteration in the strain’s cell wall. A model which integrates the roles of the cell membrane, cell wall, and divalent cations is presented. Finally, nisin resistance inL. monocytogenes ATCC 700302 conferred cross-resistance to the class IIa bacteriocin pediocin PA-1 and the class IV leuconocin S.

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Phage SPP1 Reversible Adsorption to Bacillus subtilis Cell Wall Teichoic Acids Accelerates Virus Recognition of Membrane Receptor YueB

Journal of Bacteriology ◽

10.1128/jb.00349-08 ◽

2008 ◽

Vol 190 (14) ◽

pp. 4989-4996 ◽

Cited By ~ 84

Author(s):

Catarina Baptista ◽

Mário A. Santos ◽

Carlos São-José

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Strong Dependence ◽

Teichoic Acid ◽

Wall Teichoic Acid ◽

Binding Interaction ◽

Host Cell Membrane ◽

Teichoic Acids ◽

Bacterial Surface ◽

Reversible Adsorption

ABSTRACT Bacteriophage SPP1 targets the host cell membrane protein YueB to irreversibly adsorb and infect Bacillus subtilis. Interestingly, SPP1 still binds to the surface of yueB mutants, although in a completely reversible way. We evaluated here the relevance of a reversible step in SPP1 adsorption and identified the receptor(s) involved. We show that reversible adsorption is impaired in B. subtilis mutants defective in the glucosylation pathway of teichoic acids or displaying a modified chemical composition of these polymers. The results indicate that glucosylated poly(glycerolphosphate) cell wall teichoic acid is the major target for SPP1 reversible binding. Interaction with this polymer is characterized by a fast adsorption rate showing low-temperature dependence, followed by a rapid establishment of an equilibrium state between adsorbed and free phages. This equilibrium is basically determined by the rate of phage dissociation, which exhibits a strong dependence on temperature compatible with an Arrhenius law. This allowed us to determine an activation energy of 22.6 kcal/mol for phage release. Finally, we show that SPP1 reversible interaction strongly accelerates irreversible binding to YueB. Our results support a model in which fast SPP1 adsorption to and desorption from teichoic acids allows SPP1 to scan the bacterial surface for rapid YueB recognition.

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Fine Structure of Bacillus subtilis

The Journal of Cell Biology ◽

10.1083/jcb.5.1.123 ◽

1959 ◽

Vol 5 (1) ◽

pp. 123-128 ◽

Cited By ~ 25

Author(s):

Kiyoteru Tokuyasu ◽

Eichi Yamada

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Fine Structure ◽

Cytoplasmic Membrane ◽

Tap Water ◽

Outer Part ◽

Butyl Methacrylate ◽

Smooth Structure ◽

Definition Of ◽

Made In

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO4, OsO4, or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO4 in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO4 fixation appeared to provide much better definition of the boundaries of various structures than did OsO4. With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO4 fixation) as two thin lines. In cells fixed first with OsO4 solution, and then refixed with a mixture of KMnO4 and OsO4 solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO4. One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.

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Phage SPP1 Reversible Adsorption to Bacillus subtilis Cell Wall Teichoic Acids Accelerates Virus Recognition of Membrane Receptor YueB

Journal of Bacteriology ◽

10.1128/jb.01808-08 ◽

2009 ◽

Vol 191 (5) ◽

pp. 1726-1726 ◽

Cited By ~ 1

Author(s):

Catarina Baptista ◽

Mário A. Santos ◽

Carlos São-José

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Membrane Receptor ◽

Subtilis Cell ◽

Teichoic Acids ◽

Reversible Adsorption ◽

Phage Spp1 ◽

Wall Teichoic Acids

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Proteomic Response of Bacillus subtilis to Lantibiotics Reflects Differences in Interaction with the Cytoplasmic Membrane

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01380-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5749-5757 ◽

Cited By ~ 52

Author(s):

Michaela Wenzel ◽

Bastian Kohl ◽

Daniela Münch ◽

Nadja Raatschen ◽

H. Bauke Albada ◽

...

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Pore Formation ◽

Cytoplasmic Membrane ◽

Cell Wall Biosynthesis ◽

Membrane Pore ◽

Content Type ◽

Lipid Ii ◽

Proteomic Response ◽

The Impact

ABSTRACTMersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane composition. In this study, we investigated the impact of nisin, gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization inBacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit inB. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane.

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Antibiotic-Inducible Promoter Regulated by the Cell Envelope Stress-Sensing Two-Component System LiaRS of Bacillus subtilis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.8.2888-2896.2004 ◽

2004 ◽

Vol 48 (8) ◽

pp. 2888-2896 ◽

Cited By ~ 176

Author(s):

Thorsten Mascher ◽

Sara L. Zimmer ◽

Terry-Ann Smith ◽

John D. Helmann

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Cytoplasmic Membrane ◽

Cell Envelope ◽

Inducible Promoter ◽

Two Component System ◽

Component System ◽

Lipid Ii ◽

Two Component ◽

Stress Sensing

ABSTRACT Soil bacteria are among the most prodigious producers of antibiotics. The Bacillus subtilis LiaRS (formerly YvqCE) two-component system is one of several antibiotic-sensing systems that coordinate the genetic response to cell wall-active antibiotics. Upon the addition of vancomycin or bacitracin, LiaRS autoregulates the liaIHGFSR operon. We have characterized the promoter of the lia operon and defined the cis-acting sequences necessary for antibiotic-inducible gene expression. A survey for compounds that act as inducers of the lia promoter revealed that it responds strongly to a subset of cell wall-active antibiotics that interfere with the lipid II cycle in the cytoplasmic membrane (bacitracin, nisin, ramoplanin, and vancomycin). Chemicals that perturb the cytoplasmic membrane, such as organic solvents, are also weak inducers. Thus, the reporter derived from P liaI (the liaI promoter) provides a tool for the detection and classification of antimicrobial compounds.

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Bacillus subtilis σVConfers Lysozyme Resistance by Activation of Two Cell Wall Modification Pathways, Peptidoglycan O-Acetylation and d-Alanylation of Teichoic Acids

Journal of Bacteriology ◽

10.1128/jb.06023-11 ◽

2011 ◽

Vol 193 (22) ◽

pp. 6223-6232 ◽

Cited By ~ 72

Author(s):

Veronica Guariglia-Oropeza ◽

John D. Helmann

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Double Mutant ◽

Cell Envelope ◽

Intrinsic Resistance ◽

Triple Mutant ◽

Cell Wall Modification ◽

Content Type ◽

Teichoic Acids ◽

Σ Factor

The seven extracytoplasmic function (ECF) sigma (σ) factors ofBacillus subtilisare broadly implicated in resistance to antibiotics and other cell envelope stressors mediated, in part, by regulation of cell envelope synthesis and modification enzymes. We here define the regulon of σVas including at least 20 operons, many of which are also regulated by σM, σX, or σW. The σVregulon is strongly and specifically induced by lysozyme, and this induction is key to the intrinsic resistance ofB. subtilisto lysozyme. Strains with null mutations in eithersigVor all seven ECF σ factor genes (Δ7ECF) have essentially equal increases in sensitivity to lysozyme. Induction of σVin the Δ7ECF background restores lysozyme resistance, whereas induction of σM, σX, or σWdoes not. Lysozyme resistance results from the ability of σVto activate the transcription of two operons: the autoregulatedsigV-rsiV-oatA-yrhKoperon anddltABCDE. Genetic analyses reveal thatoatAanddltare largely redundant with respect to lysozyme sensitivity: single mutants are not affected in lysozyme sensitivity, whereas anoatA dltAdouble mutant is as sensitive as asigVnull strain. Moreover, thesigV oatA dltAtriple mutant is no more sensitive than theoatA dltAdouble mutant, indicating that there are no other σV-dependent genes necessary for lysozyme resistance. Thus, we suggest that σVconfers lysozyme resistance by the activation of two cell wall modification pathways: O-acetylation of peptidoglycan catalyzed by OatA andd-alanylation of teichoic acids by DltABCDE.

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