Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors

AbstractThe bacterial cell envelope is an essential structure that protects the cell from environmental threats, while simultaneously serving as communication interface and diffusion barrier. Therefore, maintaining cell envelope integrity is of vital importance for all microorganisms. Not surprisingly, evolution has shaped conserved protection networks that connect stress perception, transmembrane signal transduction and mediation of cellular responses upon cell envelope stress. The phage shock protein (PSP) stress response is one of such conserved protection networks. Most of the knowledge about the Psp response comes from studies in the Gram-negative model bacterium, Escherichia coli where the Psp system consists of several well-defined protein components. Homologous systems were identified in representatives of Proteobacteria, Actinobacteria, and Firmicutes; however, the Psp system distribution in the microbial world remains largely unknown. By carrying out a large-scale, unbiased comparative genomics analysis, we found components of the Psp system in many bacterial and archaeal phyla and demonstrated that the PSP system deviates dramatically from the proteobacterial prototype. Two of its core proteins, PspA and PspC, have been integrated in various (often phylum-specifically) conserved protein networks during evolution. Based on protein sequence and gene neighborhood analyses of pspA and pspC homologs, we built a natural classification system of PSP networks in bacteria and archaea. We performed a comprehensive in vivo protein interaction screen for the PSP network newly identified in the Gram-positive model organism Bacillus subtilis and found a strong interconnected PSP response system, illustrating the validity of our approach. Our study highlights the diversity of PSP organization and function across many bacterial and archaeal phyla and will serve as foundation for future studies of this envelope stress response beyond model organisms.

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Swarmer cell development of the bacteriumProteus mirabilisrequires the conserved ECA biosynthesis gene,rffG

10.1101/198622 ◽

2017 ◽

Author(s):

Kristin Little ◽

Murray J. Tipping ◽

Karine A. Gibbs

Keyword(s):

Cell Morphology ◽

Stress Responses ◽

Cell Elongation ◽

Cell Envelope ◽

Membrane Composition ◽

Swarmer Cell ◽

Enterobacterial Common Antigen ◽

Envelope Stress ◽

Open Question ◽

Additional Role

AbstractIndividual cells of the bacteriumProteus mirabiliscan elongate up to 40-fold on surfaces before engaging in a cooperative surface-based motility termed swarming. How cells regulate this dramatic morphological remodeling remains an open question. In this paper, we move forward the understanding of this regulation by demonstrating thatP. mirabilisrequires the generffGfor swarmer cell elongation and subsequent swarm motility. TherffGgene encodes a protein homologous to the dTDP-glucose 4,6 dehydratase protein ofEscherichia coli, which contributes to Enterobacterial Common Antigen biosynthesis. Here we characterize therffGgene inP. mirabilis, demonstrating that it is required for the production of large lipopolysaccharide-linked moieties necessary for wild-type cell envelope integrity. We show that absence of therffGgene induces several stress-responsive pathways including those controlled by the transcriptional regulators RpoS, CaiF, and RcsB. We further show that inrffG-deficient cells, suppression of the Rcs phosphorelay, via loss of RcsB, is sufficient to induce cell elongation and swarm motility. However, loss of RcsB does not rescue cell envelope integrity defects and instead results in abnormally shaped cells, including cells producing more than two poles. We conclude that a RcsB-mediated response acts to suppress emergence of shape defects in cell envelope-compromised cells, suggesting an additional role for RcsB in maintaining cell morphology under stress conditions. We further propose that the composition of the cell envelope acts as a checkpoint before cells initiate swarmer cell elongation and motility.Importance statementP. mirabilisswarm motility has been implicated in pathogenesis. We have found that cells deploy multiple uncharacterized strategies to handle cell envelope stress beyond the Rcs phosphorelay when attempting to engage in swarm motility. While RcsB is known to directly inhibit the master transcriptional regulator for swarming, we have shown an additional role for RcsB in protecting cell morphology. These data support a growing appreciation that the Rcs phosphorelay is a multi-functional regulator of cell morphology in addition to its role in microbial stress responses. These data also strengthen the paradigm that outer membrane composition is a crucial checkpoint for modulating entry into swarm motility. Furthermore, therffG-dependent moieties provide a novel, attractive target for potential antimicrobials.

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The Rcs System in Enterobacteriaceae: Envelope Stress Responses and Virulence Regulation

Frontiers in Microbiology ◽

10.3389/fmicb.2021.627104 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jiao Meng ◽

Glenn Young ◽

Jingyu Chen

Keyword(s):

Stress Response ◽

Stress Responses ◽

Bacterial Infections ◽

Pathogenic Bacteria ◽

Cell Envelope ◽

Regulatory System ◽

Virulence Regulation ◽

Bacterial Interaction ◽

Envelope Stress

The bacterial cell envelope is a protective barrier at the frontline of bacterial interaction with the environment, and its integrity is regulated by various stress response systems. The Rcs (regulator of capsule synthesis) system, a non-orthodox two-component regulatory system (TCS) found in many members of the Enterobacteriaceae family, is one of the envelope stress response pathways. The Rcs system can sense envelope damage or defects and regulate the transcriptome to counteract stress, which is particularly important for the survival and virulence of pathogenic bacteria. In this review, we summarize the roles of the Rcs system in envelope stress responses (ESRs) and virulence regulation. We discuss the environmental and intrinsic sources of envelope stress that cause activation of the Rcs system with an emphasis on the role of RcsF in detection of envelope stress and signal transduction. Finally, the different regulation mechanisms governing the Rcs system’s control of virulence in several common pathogens are introduced. This review highlights the important role of the Rcs system in the environmental adaptation of bacteria and provides a theoretical basis for the development of new strategies for control, prevention, and treatment of bacterial infections.

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The three-component system EsrISR regulates a cell envelope stress response inCorynebacterium glutamicum

Molecular Microbiology ◽

10.1111/mmi.13839 ◽

2017 ◽

Vol 106 (5) ◽

pp. 719-741 ◽

Cited By ~ 6

Author(s):

Britta Kleine ◽

Ava Chattopadhyay ◽

Tino Polen ◽

Daniela Pinto ◽

Thorsten Mascher ◽

...

Keyword(s):

Stress Response ◽

Cell Envelope ◽

Component System ◽

Envelope Stress ◽

A Cell

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Streptococcus mutans SpxA2 relays the signal of cell envelope stress from LiaR to effectors that maintain cell wall and membrane homeostasis

Molecular Oral Microbiology ◽

10.1111/omi.12282 ◽

2020 ◽

Vol 35 (3) ◽

pp. 118-128

Author(s):

Jonathon L. Baker ◽

Sarah Saputo ◽

Roberta C. Faustoferri ◽

Robert G. Quivey

Keyword(s):

Cell Wall ◽

Streptococcus Mutans ◽

Cell Envelope ◽

Envelope Stress

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The Cell Envelope Stress Response of Bacillus subtilis towards Laspartomycin C

Antibiotics ◽

10.3390/antibiotics9110729 ◽

2020 ◽

Vol 9 (11) ◽

pp. 729

Author(s):

Angelika Diehl ◽

Thomas M. Wood ◽

Susanne Gebhard ◽

Nathaniel I. Martin ◽

Georg Fritz

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Stress Response ◽

Cell Envelope ◽

Resistance Mechanisms ◽

Detailed Knowledge ◽

Knock Out ◽

Lipid Ii ◽

Envelope Stress ◽

The Impact

Cell wall antibiotics are important tools in our fight against Gram-positive pathogens, but many strains become increasingly resistant against existing drugs. Laspartomycin C is a novel antibiotic that targets undecaprenyl phosphate (UP), a key intermediate in the lipid II cycle of cell wall biosynthesis. While laspartomycin C has been thoroughly examined biochemically, detailed knowledge about potential resistance mechanisms in bacteria is lacking. Here, we use reporter strains to monitor the activity of central resistance modules in the Bacillus subtilis cell envelope stress response network during laspartomycin C attack and determine the impact on the resistance of these modules using knock-out strains. In contrast to the closely related UP-binding antibiotic friulimicin B, which only activates ECF σ factor-controlled stress response modules, we find that laspartomycin C additionally triggers activation of stress response systems reacting to membrane perturbation and blockage of other lipid II cycle intermediates. Interestingly, none of the studied resistance genes conferred any kind of protection against laspartomycin C. While this appears promising for therapeutic use of laspartomycin C, it raises concerns that existing cell envelope stress response networks may already be poised for spontaneous development of resistance during prolonged or repeated exposure to this new antibiotic.

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A Periplasmic LolA Derivative with a Lethal Disulfide Bond Activates the Cpx Stress Response System

Journal of Bacteriology ◽

10.1128/jb.00821-10 ◽

2010 ◽

Vol 192 (21) ◽

pp. 5657-5662 ◽

Cited By ~ 11

Author(s):

Kazuyuki Tao ◽

Shoji Watanabe ◽

Shin-ichiro Narita ◽

Hajime Tokuda

Keyword(s):

Disulfide Bond ◽

Cell Envelope ◽

Two Component System ◽

Outer Membranes ◽

Inhibition Of Growth ◽

Atp Binding Cassette Transporter ◽

Intramolecular Disulfide Bond ◽

Envelope Stress ◽

Acyl Chains ◽

Stress Response System

ABSTRACT LolA accommodates the acyl chains of lipoproteins in its hydrophobic cavity and shuttles between the inner and outer membranes through the hydrophilic periplasm to place lipoproteins in the outer membrane. The LolA(I93C/F140C) derivative, in which Cys replaces Ile at position 93 and Phe at position 140, strongly inhibited growth in the absence of a reducing agent because of the lethal intramolecular disulfide bond between the two Cys residues. Expression of I93C/F140C was found to activate the Cpx two-component system, which responds to cell envelope stress. The inhibition of growth by I93C/F140C was partly suppressed by overproduction of LolCDE, which is an ATP-binding cassette transporter and mediates the transfer of lipoproteins from the inner membrane to LolA. A substantial portion of the oxidized form, but not the reduced one, of I93C/F140C expressed on LolCDE overproduction was recovered in the membrane fraction, whereas wild-type LolA was localized in the periplasm even when LolCDE was overproduced. Moreover, LolCDE overproduction stabilized I93C/F140C and therefore caused an increase in its level. Taken together, these results indicate that oxidized I93C/F140C stably binds to LolCDE, which causes strong envelope stress.

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