Mining marine metagenomes revealed a quorum-quenching lactonase with improved biochemical properties that inhibits the food spoilage bacteria Pseudomonas fluorescens

2021 ◽  
Author(s):  
Rami Haramati ◽  
Shlomit Dor ◽  
David Gurevich ◽  
Doron Levy ◽  
Dekel Freund ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Marine Environment ◽  
Recombinant Expression ◽  
Cell Communication ◽  
Bacterial Pathogen ◽  
Quorum Quenching ◽  
Amino Acid Identity ◽  
Biochemical Properties ◽  
Food Spoilage ◽  
Spoilage Bacteria

The marine environment presents great potential as a source of microorganisms that possess novel enzymes with unique activities and biochemical properties. Examples of such are the quorum-quenching (QQ) enzymes that hydrolyze bacterial quorum-sensing (QS) signaling molecules, such as N-acyl-homoserine lactones (AHLs). QS is a form of cell-to-cell communication that enables bacteria to synchronize gene expression in correlation with population density. Searching marine metagenomes for sequences homologous to an AHL lactonase from the phosphotriesterase-like lactonase (PLL) family, we identified new putative AHL lactonases (sharing 30-40% amino acid identity to a thermostable PLL member). Phylogenetic analysis indicated that these putative AHL lactonases comprise a new clade of marine enzymes in the PLL family. Following recombinant expression and purification, we verified the AHL lactonase activity for one of these proteins, named marine originated Lactonase Related Protein (moLRP). This enzyme presented greater activity and stability at a broad range of temperatures and pH, and tolerance to high salinity levels (up to 5M NaCl), as well as higher durability in bacterial culture, compared to another PLL member. The addition of purified moLRP to cultures of Pseudomonas fluorescens inhibited its extracellular protease activity, expression of the protease encoding gene, biofilm formation, and the sedimentation process in milk-based medium. These findings suggest that moLRP is adapted to the marine environment, and can potentially serve as an effective QQ enzyme, inhibiting the QS process in gram-negative bacteria involved in food spoilage. Importance Our results emphasize the potential of sequence and structure-based identification of new quorum-quenching (QQ) enzymes from environmental metagenomes, such as from the ocean, with improved stability or activity. The findings also suggest that purified QQ enzymes can present new strategies against food spoilage, in addition to their recognized involvement in inhibiting bacterial pathogen virulence factors. Future studies on the delivery and safety of enzymatic QQ strategy against bacterial food spoilage should be performed.

scholarly journals Quantification of anti-quorum sensing and antibiofilm activity of Phyllosphere bacteria against food spoilage bacteria

2021 ◽  
pp. e919
Author(s):  
Valencia Vanessa ◽  
Diana Elizabeth Waturangi
Keyword(s):  
Quorum Sensing ◽  
Bioactive Compounds ◽  
Large Population ◽  
Cell Communication ◽  
Antibiofilm Activity ◽  
Food Spoilage ◽  
Biofilm Inhibition ◽  
Sensing Mechanism ◽  
Spoilage Bacteria ◽  
Phyllosphere Bacteria

Food spoilage and microbial contamination require  attention during the food production process since the presence of these bacteria can create problems including the formation of biofilms produced by these  bacteria. Biofilm formations are initiated through cell-to-cell communication which is called quorum sensing mechanism. Hence, inhibition of this communication  mechanism could be one of the solutions to inhibit  biofilm formation. Therefore, exploration of bioactive compounds from various sources including  hyllosphere bacteria with anti-quorum sensing inhibition activities is important. Phyllosphere bacteria are a community of bacteria found on the surface of plant leaves at a very  large population. These bacteria can produce bioactive compounds that can inhibit quorum sensing mechanism. In this study, 54 phyllosphere bacteria  isolates were tested, 8 bacterial isolates had potential effect to inhibit quorum sensing. From biofilm inhibition assay, the highest percentages were showed by  ifferent phyllosphere isolates against each pathogen. Whereas, for biofilm destruction assay, JB 8F isolate had the highest percentage of destruction biofilm activity  against biofilm formed by Bacillus cereus and  Shewanella putrefaciens. Eight isolates of phyllosphere  bacteria had the potential as quorum quencher and  anti-biofilm agents, both for inhibition and destruction of biofilm.  

Investigation of the roles of AgrA and σB regulators in Listeria monocytogenes adaptation to roots and soil

2020 ◽  
Vol 367 (3) ◽  
Author(s):  
Catarina M Marinho ◽  
Dominique Garmyn ◽  
Laurent Gal ◽  
Maja Z Brunhede ◽  
Conor O'Byrne ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Parental Strain ◽  
Festuca Arundinacea ◽  
Double Mutant ◽  
Response Regulator ◽  
Cell Communication ◽  
Bacterial Pathogen ◽  
Transcriptional Analysis ◽  
Root Colonisation ◽  
Two Systems

ABSTRACT Little is known about the regulatory mechanisms that ensure the survival of the food-borne bacterial pathogen Listeria monocytogenes in the telluric environment and on roots. Earlier studies have suggested a regulatory overlap between the Agr cell–cell communication system and the general stress response regulator σB. Here, we investigated the contribution of these two systems to root colonisation and survival in sterilised and biotic soil. The ability to colonise the roots of the grass Festuca arundinacea was significantly compromised in the double mutant (∆agrA∆sigB). In sterile soil at 25°C, a significant defect was observed in the double mutant, suggesting some synergy between these systems. However, growth was observed and similar population dynamics were shown in the parental strain, ΔagrA and ΔsigB mutants. In biotic soil at 25°C, viability of the parental strain declined steadily over a two-week period highlighting the challenging nature of live soil environments. Inactivation of the two systems further decreased survival. The synergistic effect of Agr and σB was stronger in biotic soil. Transcriptional analysis confirmed the expected effects of the mutations on known Agr- and σB-dependent genes. Data highlight the important role that these global regulatory systems play in the natural ecology of this pathogen.

scholarly journals Identification of FadT as a Novel Quorum Quenching Enzyme for the Degradation of Diffusible Signal Factor in Cupriavidus pinatubonensis Strain HN-2

2021 ◽  
Vol 22 (18) ◽  
pp. 9862
Author(s):  
Xudan Xu ◽  
Tian Ye ◽  
Wenping Zhang ◽  
Tian Zhou ◽  
Xiaofan Zhou ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Xanthomonas Campestris ◽  
Cell Communication ◽  
Maximal Activity ◽  
Quorum Quenching ◽  
Site Directed Mutagenesis ◽  
Diffusible Signal Factor ◽  
High Enzymatic Activity ◽  
Xanthomonas Campestris Pv Campestris ◽  
Potential Use

Quorum sensing (QS) is a microbial cell–cell communication mechanism and plays an important role in bacterial infections. QS-mediated bacterial infections can be blocked through quorum quenching (QQ), which hampers signal accumulation, recognition, and communication. The pathogenicity of numerous bacteria, including Xanthomonas campestris pv. campestris (Xcc), is regulated by diffusible signal factor (DSF), a well-known fatty acid signaling molecule of QS. Cupriavidus pinatubonensis HN-2 could substantially attenuate the infection of XCC through QQ by degrading DSF. The QQ mechanism in strain HN-2, on the other hand, is yet to be known. To understand the molecular mechanism of QQ in strain HN-2, we used whole-genome sequencing and comparative genomics studies. We discovered that the fadT gene encodes acyl-CoA dehydrogenase as a novel QQ enzyme. The results of site-directed mutagenesis demonstrated the requirement of fadT gene for DSF degradation in strain HN-2. Purified FadT exhibited high enzymatic activity and outstanding stability over a broad pH and temperature range with maximal activity at pH 7.0 and 35 °C. No cofactors were required for FadT enzyme activity. The enzyme showed a strong ability to degrade DSF. Furthermore, the expression of fadT in Xcc results in a significant reduction in the pathogenicity in host plants, such as Chinese cabbage, radish, and pakchoi. Taken together, our results identified a novel DSF-degrading enzyme, FadT, in C. pinatubonensis HN-2, which suggests its potential use in the biological control of DSF-mediated pathogens.

scholarly journals Improving the production of AHL lactonase AiiO-AIO6 from Ochrobactrum sp. M231 in intracellular protease-deficientBacillus subtilis

2020 ◽  
Author(s):  
Rui Xia ◽  
Yalin Yang ◽  
Xingliang Pan ◽  
Chenchen Gao ◽  
Yuanyuan Yao ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Positive Impact ◽  
Cell Communication ◽  
Quorum Quenching ◽  
Catalytic Triad ◽  
Heterologous Proteins ◽  
Extracellular Production ◽  
Expression Plasmid ◽  
Encoding Genes ◽  
Hydrolase Family

Abstract Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway.To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis1A751 were constructed by individually knocking out the intracellular protease-encoding genes (tepA, ymfH, yrrNandywpE). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔtepA, BSΔymfH, BSΔyrrNand BSΔywpE) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔywpE in shake flask reached 1416.47 U/mL/OD600, which was about 121% higher than that of the wild-type strain. Furthermore, LC-MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6was classified as a member of the α/β hydrolase family with a conserved “nucleophile-acid-histidine” catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

scholarly journals Bacterial Quorum-Quenching Lactonase Hydrolyzes Fungal Mycotoxin and Reduces Pathogenicity of Penicillium expansum—Suggesting a Mechanism of Bacterial Antagonism

2021 ◽  
Vol 7 (10) ◽  
pp. 826
Author(s):  
Shlomit Dor ◽  
Dov Prusky ◽  
Livnat Afriat-Jurnou
Keyword(s):  
Cell Wall ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Molecular Mechanisms ◽  
Recombinant Expression ◽  
Bacterial Species ◽  
Quorum Quenching ◽  
Host Cells ◽  
Penicillium Expansum ◽  
Fungal Colonization

Penicillium expansum is a necrotrophic wound fungal pathogen that secrets virulence factors to kill host cells including cell wall degrading enzymes (CWDEs), proteases, and mycotoxins such as patulin. During the interaction between P. expansum and its fruit host, these virulence factors are strictly modulated by intrinsic regulators and extrinsic environmental factors. In recent years, there has been a rapid increase in research on the molecular mechanisms of pathogenicity in P. expansum; however, less is known regarding the bacteria–fungal communication in the fruit environment that may affect pathogenicity. Many bacterial species use quorum-sensing (QS), a population density-dependent regulatory mechanism, to modulate the secretion of quorum-sensing signaling molecules (QSMs) as a method to control pathogenicity. N-acyl homoserine lactones (AHLs) are Gram-negative QSMs. Therefore, QS is considered an antivirulence target, and enzymes degrading these QSMs, named quorum-quenching enzymes, have potential antimicrobial properties. Here, we demonstrate that a bacterial AHL lactonase can also efficiently degrade a fungal mycotoxin. The mycotoxin is a lactone, patulin secreted by fungi such as P. expansum. The bacterial lactonase hydrolyzed patulin at high catalytic efficiency, with a kcat value of 0.724 ± 0.077 s−1 and KM value of 116 ± 33.98 μM. The calculated specific activity (kcat/KM) showed a value of 6.21 × 103 s−1M−1. While the incubation of P. expansum spores with the purified lactonase did not inhibit spore germination, it inhibited colonization by the pathogen in apples. Furthermore, adding the purified enzyme to P. expansum culture before infecting apples resulted in reduced expression of genes involved in patulin biosynthesis and fungal cell wall biosynthesis. Some AHL-secreting bacteria also express AHL lactonase. Here, phylogenetic and structural analysis was used to identify putative lactonase in P. expansum. Furthermore, following recombinant expression and purification of the newly identified fungal enzyme, its activity with patulin was verified. These results indicate a possible role for patulin and lactonases in inter-kingdom communication between fungi and bacteria involved in fungal colonization and antagonism and suggest that QQ lactonases can be used as potential antifungal post-harvest treatment.

Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F

1993 ◽  
Vol 13 (12) ◽  
pp. 7802-7812
Author(s):  
M Ivey-Hoyle ◽  
R Conroy ◽  
H E Huber ◽  
P J Goodhart ◽  
A Oliff ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Hela Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Identity ◽  
Biochemical Properties ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Novel Protein

E2F is a mammalian transcription factor that appears to play an important role in cell cycle regulation. While at least two proteins (E2F-1 and DP-1) with E2F-like activity have been cloned, studies from several laboratories suggest that additional homologs may exist. A novel protein with E2F-like properties, designated E2F-2, was cloned by screening a HeLa cDNA library with a DNA probe derived from the DNA binding domain of E2F-1 (K. Helin, J. A. Lees, M. Vidal, N. Dyson, E. Harlow, and A. Fattaey, Cell 70:337-350, 1992). E2F-2 exhibits overall 46% amino acid identity to E2F-1. Both the sequence and the function of the DNA and retinoblastoma gene product binding domains of E2F-1 are conserved in E2F-2. The DNA binding activity of E2F-2 is dramatically enhanced by complementation with particular sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified components of HeLa cell E2F, and anti-E2F-2 antibodies cross-react with components of purified HeLa cell E2F. These observations are consistent with a model in which E2F binds DNA as a heterodimer of two distinct proteins, and E2F-2 is functionally and immunologically related to one of these proteins.

Sign in / Sign up

Export Citation Format

Share Document