scholarly journals Reversible regulation of conjugation of Bacillus subtilis plasmid pLS20 by the quorum sensing peptide responsive anti-repressor RappLS20

2020 ◽  
Vol 48 (19) ◽  
pp. 10785-10801
Author(s):  
Praveen K Singh ◽  
Ester Serrano ◽  
Gayetri Ramachandran ◽  
Andrés Miguel-Arribas ◽  
César Gago-Cordoba ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Bacillus Subtilis ◽  
Quorum Sensing ◽  
Regulatory Genes ◽  
Dna Looping ◽  
Regulatory Proteins ◽  
Bacterial Communication ◽  
Health Related ◽  
Operator Site

Abstract Quorum sensing plays crucial roles in bacterial communication including in the process of conjugation, which has large economical and health-related impacts by spreading antibiotic resistance. The conjugative Bacillus subtilis plasmid pLS20 uses quorum sensing to determine when to activate the conjugation genes. The main conjugation promoter, Pc, is by default repressed by a regulator RcopLS20 involving DNA looping. A plasmid-encoded signalling peptide, Phr*pLS20, inactivates the anti-repressor of RcopLS20, named RappLS20, which belongs to the large group of RRNPP family of regulatory proteins. Here we show that DNA looping occurs through interactions between two RcopLS20 tetramers, each bound to an operator site. We determined the relative promoter strengths for all the promoters involved in synthesizing the regulatory proteins of the conjugation genes, and constructed an in vivo system uncoupling these regulatory genes to show that RappLS20 is sufficient for activating conjugation in vivo. We also show that RappLS20 actively detaches RcopLS20 from DNA by preferentially acting on the RcopLS20 molecules involved in DNA looping, resulting in sequestration but not inactivation of RcopLS20. Finally, results presented here in combination with our previous results show that activation of conjugation inhibits competence and competence development inhibits conjugation, indicating that both processes are mutually exclusive.

scholarly journals Anti-quorum Sensing and Protective Efficacies of Naringin Against Aeromonas hydrophila Infection in Danio rerio

2020 ◽  
Vol 11 ◽  
Author(s):  
Ramanathan Srinivasan ◽  
Kannan Rama Devi ◽  
Sivasubramanian Santhakumari ◽  
Arunachalam Kannappan ◽  
Xiaomeng Chen ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Quorum Sensing ◽  
Aeromonas Hydrophila ◽  
Biofilm Formation ◽  
Bacterial Load ◽  
Antibiotic Resistance Genes ◽  
Alternative Methods ◽  
Zebrafish Model ◽  
Virulence Gene Expression

It is now well known that the quorum sensing (QS) mechanism coordinates the production of several virulence factors and biofilm formation in most pathogenic microorganisms. Aeromonas hydrophila is a prime pathogen responsible for frequent outbreaks in aquaculture settings. Recent studies have also continuously reported that A. hydrophila regulates virulence factor production and biofilm formation through the QS system. In addition to the presence of antibiotic resistance genes, biofilm-mediated antibiotic resistance increases the severity of A. hydrophila infections. To control the bacterial pathogenesis and subsequent infections, targeting the QS mechanism has become one of the best alternative methods. Though very few compounds were identified as QS inhibitors against A. hydrophila, to date, the screening and identification of new and effective natural QS inhibitors is a dire necessity to control the infectious A. hydrophila. The present study endorses naringin (NA) as an anti-QS and anti-infective agent against A. hydrophila. Initially, the NA showed a concentration-dependent biofilm reduction against A. hydrophila. Furthermore, the results of microscopic analyses and quantitative virulence assays displayed the promise of NA as a potential anti-QS agent. Subsequently, the downregulation of ahh1, aerA, lip and ahyB validate the interference of NA in virulence gene expression. Furthermore, the in vivo assays were carried out in zebrafish model system to evaluate the anti-infective potential of NA. The outcome of the immersion challenge assay showed that the recovery rate of the zebrafish has substantially increased upon treatment with NA. Furthermore, the quantification of the bacterial load upon NA treatment showed a decreased level of bacterial counts in zebrafish when compared to the untreated control. Moreover, the NA treatment averts the pathogen-induced histoarchitecture damages in vital organs of zebrafish, compared to their respective controls. The current study has thus analyzed the anti-QS and anti-infective capabilities of NA and could be employed to formulate effective treatment measures against A. hydrophila infections.

scholarly journals Regulators of the Bacillus subtilis cydABCD Operon: Identification of a Negative Regulator, CcpA, and a Positive Regulator, ResD

2007 ◽  
Vol 189 (9) ◽  
pp. 3348-3358 ◽  
Author(s):  
Ankita Puri-Taneja ◽  
Matthew Schau ◽  
Yinghua Chen ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Negative Regulator ◽  
Regulatory Proteins ◽  
Regulation Of Respiration ◽  
Regulator Protein ◽  
Promoter Fusion

ABSTRACT The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions −58 and −107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3′-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions −4 to −33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.

scholarly journals The quorum sensing inhibitor furanone C-30 rapidly loses its tobramycin potentiating activity against Pseudomonas aeruginosa biofilms during experimental evolution

2021 ◽  
Author(s):  
Mona Bové ◽  
Xuerui Bao ◽  
Andrea Sass ◽  
Aurélie Crabbé ◽  
Tom Coenye
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Quorum Sensing ◽  
Experimental Evolution ◽  
Aminoglycoside Antibiotic ◽  
Sequencing Analysis ◽  
Cystic Fibrosis Sputum ◽  
Quorum Sensing Inhibitors ◽  
C 30

The use of quorum sensing inhibitors (QSI) has been proposed as an alternative strategy to combat antibiotic resistance. QSI reduce the virulence of a pathogen without killing it and it is claimed that resistance to such compounds is less likely to develop although there is a lack of experimental data supporting this hypothesis. Additionally, such studies are often carried out in conditions that do not mimic the in vivo situation. In the present study, we evaluated whether a combination of the QSI furanone C-30 and the aminoglycoside antibiotic tobramycin is ‘evolution-proof’ when it is used to eradicate Pseudomonas aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium. We found that the biofilm eradicating activity of the tobramycin/furanone C-30 combination decreased already after 5 treatment cycles. The antimicrobial susceptibility of P. aeruginosa to tobramycin decreased 8-fold after 16 cycles of treatment with the tobramycin/furanone C-30 combination. Furthermore, microcalorimetry revealed changes in the metabolic activity of P. aeruginosa exposed to furanone C-30, tobramycin, and the combination. Whole-genome sequencing analysis of the evolved strains exposed to the combination identified mutations in mexT, fusA1 and parS, genes known to be involved in antibiotic resistance. In P. aeruginosa treated with furanone C-30 alone, a deletion in mexT was also observed. Our data indicate that furanone C-30 is not ‘evolution-proof’ and quickly becomes ineffective as a tobramycin potentiator.

scholarly journals Metabolic Exchange and Energetic Coupling between Nutritionally Stressed Bacterial Species: Role of Quorum-Sensing Molecules

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David Ranava ◽  
Cassandra Backes ◽  
Ganesan Karthikeyan ◽  
Olivier Ouari ◽  
Audrey Soric ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Bacterial Species ◽  
Emergent Properties ◽  
Desulfovibrio Vulgaris ◽  
Interspecies Interactions ◽  
Content Type ◽  
Bacterial Communication ◽  
Metabolic Exchange

ABSTRACT Formation of multispecies communities allows nearly every niche on earth to be colonized, and the exchange of molecular information among neighboring bacteria in such communities is key for bacterial success. To clarify the principles controlling interspecies interactions, we previously developed a coculture model with two anaerobic bacteria, Clostridium acetobutylicum (Gram positive) and Desulfovibrio vulgaris Hildenborough (Gram negative, sulfate reducing). Under conditions of nutritional stress for D. vulgaris, the existence of tight cell-cell interactions between the two bacteria induced emergent properties. Here, we show that the direct exchange of carbon metabolites produced by C. acetobutylicum allows D vulgaris to duplicate its DNA and to be energetically viable even without its substrates. We identify the molecular basis of the physical interactions and how autoinducer-2 (AI-2) molecules control the interactions and metabolite exchanges between C. acetobutylicum and D. vulgaris (or Escherichia coli and D. vulgaris). With nutrients, D. vulgaris produces a small molecule that inhibits in vitro the AI-2 activity and could act as an antagonist in vivo. Sensing of AI-2 by D. vulgaris could induce formation of an intercellular structure that allows directly or indirectly metabolic exchange and energetic coupling between the two bacteria. IMPORTANCE Bacteria have usually been studied in single culture in rich media or under specific starvation conditions. However, in nature they coexist with other microorganisms and build an advanced society. The molecular bases of the interactions controlling this society are poorly understood. Use of a synthetic consortium and reducing complexity allow us to shed light on the bacterial communication at the molecular level. This study presents evidence that quorum-sensing molecule AI-2 allows physical and metabolic interactions in the synthetic consortium and provides new insights into the link between metabolism and bacterial communication.

scholarly journals Assessing the Molecular Targets and Mode of Action of Furanone C-30 on Pseudomonas aeruginosa Quorum Sensing

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1620
Author(s):  
Victor Markus ◽  
Karina Golberg ◽  
Kerem Teralı ◽  
Nazmi Ozer ◽  
Esti Kramarsky-Winter ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Quorum Sensing ◽  
Conformational Changes ◽  
Mode Of Action ◽  
Pathogenic Bacteria ◽  
Molecular Targets ◽  
Resistant Bacteria ◽  
Public Healthcare ◽  
C 30

Quorum sensing (QS), a sophisticated system of bacterial communication that depends on population density, is employed by many pathogenic bacteria to regulate virulence. In view of the current reality of antibiotic resistance, it is expected that interfering with QS can address bacterial pathogenicity without stimulating the incidence of resistance. Thus, harnessing QS inhibitors has been considered a promising approach to overriding bacterial infections and combating antibiotic resistance that has become a major threat to public healthcare around the globe. Pseudomonas aeruginosa is one of the most frequent multidrug-resistant bacteria that utilize QS to control virulence. Many natural compounds, including furanones, have demonstrated strong inhibitory effects on several pathogens via blocking or attenuating QS. While the natural furanones show no activity against P. aeruginosa, furanone C-30, a brominated derivative of natural furanone compounds, has been reported to be a potent inhibitor of the QS system of the notorious opportunistic pathogen. In the present study, we assess the molecular targets and mode of action of furanone C-30 on P. aeruginosa QS system. Our results suggest that furanone C-30 binds to LasR at the ligand-binding site but fails to establish interactions with the residues crucial for the protein’s productive conformational changes and folding, thus rendering the protein dysfunctional. We also show that furanone C-30 inhibits RhlR, independent of LasR, suggesting a complex mechanism for the agent beyond what is known to date.

scholarly journals Interaction of Acinetobacter baumannii with Human Serum Albumin: Does the Host Determines the Outcome?

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 833
Author(s):  
Camila Pimentel ◽  
Casin Le ◽  
Marisel R. Tuttobene ◽  
Tomas Subils ◽  
Krisztina M. Papp-Wallace ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Human Serum Albumin ◽  
Quorum Sensing ◽  
Serum Albumin ◽  
Human Serum ◽  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Model Systems ◽  
Expression Levels ◽  
Multiple Drugs

Acinetobacter baumannii has become a serious threat to human health due to its extreme antibiotic resistance, environmental persistence, and capacity to survive within the host. Two A. baumannii strains, A118 and AB5075, commonly used as model systems, and three carbapenem-resistant strains, which are becoming ever more dangerous due to the multiple drugs they can resist, were exposed to 3.5% human serum albumin (HSA) and human serum (HS) to evaluate their response with respect to antimicrobial resistance, biofilm formation, and quorum sensing, all features responsible for increasing survival and persistence in the environment and human body. Expression levels of antibiotic resistance genes were modified differently when examined in different strains. The cmlA gene was upregulated or downregulated in conditions of exposure to 3.5% HSA or HS depending on the strain. Expression levels of pbp1 and pbp3 tended to be increased by the presence of HSA and HS, but the effect was not seen in all strains. A. baumannii A118 growing in the presence of HS did not experience increased expression of these genes. Aminoglycoside-modifying enzymes were also expressed at higher or lower levels in the presence of HSA or HS. Still, the response was not uniform; in some cases, expression was enhanced, and in other cases, it was tapered. While A. baumannii AB5075 became more susceptible to rifampicin in the presence of 3.5% HSA or HS, strain A118 did not show any changes. Expression of arr2, a gene involved in resistance to rifampicin present in A. baumannii AMA16, was expressed at higher levels when HS was present in the culture medium. HSA and HS reduced biofilm formation and production of N-Acyl Homoserine Lactone, a compound intimately associated with quorum sensing. In conclusion, HSA, the main component of HS, stimulates a variety of adaptative responses in infecting A. baumannii strains.

scholarly journals Peptide signaling without feedback in signal production operates as a true quorum sensing communication system in Bacillus subtilis

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Iztok Dogsa ◽  
Mihael Spacapan ◽  
Anna Dragoš ◽  
Tjaša Danevčič ◽  
Žiga Pandur ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Quorum Sensing ◽  
Cell Density ◽  
Communication System ◽  
Communication Systems ◽  
Feedback Loop ◽  
Signal Molecules ◽  
Specific Cell ◽  
Sharp Transition

AbstractBacterial quorum sensing (QS) is based on signal molecules (SM), which increase in concentration with cell density. At critical SM concentration, a variety of adaptive genes sharply change their expression from basic level to maximum level. In general, this sharp transition, a hallmark of true QS, requires an SM dependent positive feedback loop, where SM enhances its own production. Some communication systems, like the peptide SM-based ComQXPA communication system of Bacillus subtilis, do not have this feedback loop and we do not understand how and if the sharp transition in gene expression is achieved. Based on experiments and mathematical modeling, we observed that the SM peptide ComX encodes the information about cell density, specific cell growth rate, and even oxygen concentration, which ensure power-law increase in SM production. This enables together with the cooperative response to SM (ComX) a sharp transition in gene expression level and this without the SM dependent feedback loop. Due to its ultra-sensitive nature, the ComQXPA can operate at SM concentrations that are 100–1000 times lower than typically found in other QS systems, thereby substantially reducing the total metabolic cost of otherwise expensive ComX peptide.

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