Xanthomonas campestris RpfB is a fatty Acyl-CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase

Diffusible signal factors (DSFs) are medium-chain fatty acids that induce bacterial quorum sensing. Among these compounds, BDSF is a structural analog of DSF that is commonly detected in bacterial species (e.g., Xanthomonas, Pseudomonas, and Burkholderia). Additionally, BDSF contributes to the interkingdom communication regulating fungal life stage transitions. How BDSF is sensed in Xanthomonas spp. and the functional diversity between BDSF and DSF remain unclear. In this study, we generated genetic and biochemical evidence that BDSF is a low-active regulator of X. campestris pv. campestris quorum sensing, whereas trans-BDSF seems not a signaling compound. BDSF is detected by the sensor histidine kinase RpfC. Although BDSF has relatively low physiological activities, it binds to the RpfC sensor with a high affinity and activates RpfC autophosphorylation to a level that is similar to that induced by DSF in vitro. The inconsistency in the physiological and biochemical activities of BDSF is not due to RpfC–RpfG phosphorylation or RpfG hydrolase. Neither BDSF nor DSF controls the phosphotransferase and phosphatase activities of RpfC or the ability of RpfG hydrolase to degrade the bacterial second messenger cyclic di-GMP. We demonstrated that BDSF is prone to degradation by RpfB, a critical fatty acyl-CoA ligase involved in the turnover of DSF-family signals. rpfB mutations lead to substantial increases in BDSF-induced quorum sensing. Although DSF and BDSF are similarly detected by RpfC, our data suggest that their differential degradation in cells is the major factor responsible for the diversity in their physiological effects.

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Xanthomonas campestris Promotes Diffusible Signal Factor Biosynthesis and Pathogenicity by Utilizing Glucose and Sucrose from Host Plants

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-18-0187-r ◽

2019 ◽

Vol 32 (2) ◽

pp. 157-166 ◽

Cited By ~ 1

Author(s):

Chunyan Zhang ◽

Mingfa Lv ◽

Wenfang Yin ◽

Tingyan Dong ◽

Changqing Chang ◽

...

Keyword(s):

Host Plant ◽

Xanthomonas Campestris ◽

Infection Process ◽

Plant Metabolites ◽

Multiple Strategies ◽

Exogenous Addition ◽

Diffusible Signal Factor ◽

Signal Production ◽

Induced Changes ◽

Encoding Genes

The plant pathogen Xanthomonas campestris pv. campestris produces diffusible signal factor (DSF) quorum sensing (QS) signals to regulate its biological functions and virulence. Our previous study showed that X. campestris pv. campestris utilizes host plant metabolites to enhance the biosynthesis of DSF family signals. However, it is unclear how X. campestris pv. campestris benefits from the metabolic products of the host plant. In this study, we observed that the host plant metabolites not only boosted the production of the DSF family signals but also modulated the expression levels of DSF-regulated genes in X. campestris pv. campestris. Infection with X. campestris pv. campestris induced changes in the expression of many sugar transporter genes in Arabidopsis thaliana. Exogenous addition of sucrose or glucose, which are the major products of photosynthesis in plants, enhanced DSF signal production and X. campestris pv. campestris pathogenicity in the Arabidopsis model. In addition, several sucrose hydrolase–encoding genes in X. campestris pv. campestris and sucrose invertase–encoding genes in the host plant were notably upregulated during the infection process. These enzymes hydrolyzed sucrose to glucose and fructose, and in trans expression of one of these enzymes, CINV1 of A. thaliana or XC_0805 of X. campestris pv. campestris, enhanced DSF signal biosynthesis in X. campestris pv. campestris in the presence of sucrose. Taken together, our findings demonstrate that X. campestris pv. campestris applies multiple strategies to utilize host plant sugars to enhance QS and pathogenicity.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22189862 ◽

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.

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The cis -2-dodecenoic acid (BDSF) quorum sensing system in Burkholderia cenocepacia

Applied and Environmental Microbiology ◽

10.1128/aem.02342-21 ◽

2022 ◽

Author(s):

Mingfang Wang ◽

Xia Li ◽

Shihao Song ◽

Chaoyu Cui ◽

Lian-Hui Zhang ◽

...

Keyword(s):

Quorum Sensing ◽

Bacterial Infections ◽

Xanthomonas Campestris ◽

Cell Communication ◽

Burkholderia Cenocepacia ◽

Bacterial Cells ◽

Communication Signals ◽

Signal Perception ◽

Sensing System ◽

Diffusible Signal Factor

It has been demonstrated that quorum sensing (QS) is widely employed by bacterial cells to coordinately regulate various group behaviors. Diffusible signal factor (DSF)-type signals have emerged as a growing family of conserved cell-cell communication signals. In addition to the DSF signal initially identified in Xanthomonas campestris pv. campestris, B urkholderia d iffusible s ignal f actor (BDSF, cis -2-dodecenoic acid) has been recognized as a conserved DSF-type signal with specific characteristics in both signal perception and transduction from DSF signals. Here, we review the history and current progress of the research of this type of signal, especially focusing on its biosynthesis, signaling pathways, and biological functions. We also discuss and explore the huge potential of targeting this kind of QS system as a new therapeutic strategy to control bacterial infections and diseases.

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Active Multienzyme Assemblies for Long-Chain Olefinic Hydrocarbon Biosynthesis

Journal of Bacteriology ◽

10.1128/jb.00890-16 ◽

2017 ◽

Vol 199 (9) ◽

Cited By ~ 12

Author(s):

James K. Christenson ◽

Matthew R. Jensen ◽

Brandon R. Goblirsch ◽

Fatuma Mohamed ◽

Wei Zhang ◽

...

Keyword(s):

Active Sites ◽

Xanthomonas Campestris ◽

Fatty Acyl ◽

Biosynthetic Activity ◽

Content Type ◽

Large Structures ◽

Negative Stain ◽

Acyl Coenzyme A ◽

Hydrocarbon Biosynthesis ◽

Long Chain

ABSTRACT Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from Xanthomonas campestris in Escherichia coli and purification over His6 and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive β-lactone intermediate produced by the OleC-catalyzed reaction. IMPORTANCE Bacteria biosynthesize hydrophobic molecules to maintain a membrane, store carbon, and for antibiotics that help them survive in their niche. The hydrophobic compounds are often synthesized by a multidomain protein or by large multienzyme assemblies. The present study reports on the discovery that long-chain olefinic hydrocarbons made by bacteria from different phyla are produced by multienzyme assemblies in X. campestris. The OleBCD multienzyme assemblies are thought to compartmentalize and sequester olefin biosynthesis from the rest of the cell. This system provides additional insights into how bacteria control specific biosynthetic pathways.

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Diffusible Signal Factor-Dependent Cell-Cell Signaling and Virulence in the Nosocomial Pathogen Stenotrophomonas maltophilia

Journal of Bacteriology ◽

10.1128/jb.00310-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4964-4968 ◽

Cited By ~ 100

Author(s):

Yvonne Fouhy ◽

Karl Scanlon ◽

Katherine Schouest ◽

Charles Spillane ◽

Lisa Crossman ◽

...

Keyword(s):

Antibiotic Resistance ◽

Cell Signaling ◽

Stenotrophomonas Maltophilia ◽

Xanthomonas Campestris ◽

Signaling System ◽

Nosocomial Pathogen ◽

Diffusible Signal Factor ◽

Dependent Cell ◽

A Cell ◽

Cell Cell

ABSTRACT The genome of Stenotrophomonas maltophilia encodes a cell-cell signaling system that is highly related to the diffusible signal factor (DSF)-dependent system of the phytopathogen Xanthomonas campestris. Here we show that in S. maltophilia, DSF signaling controls factors contributing to the virulence and antibiotic resistance of this important nosocomial pathogen.

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Expression of the gum Operon Directing Xanthan Biosynthesis in Xanthomonas campestris and Its Regulation In Planta

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.6.768 ◽

2001 ◽

Vol 14 (6) ◽

pp. 768-774 ◽

Cited By ~ 51

Author(s):

Adrian A. Vojnov ◽

Holly Slater ◽

Michael J. Daniels ◽

J. Maxwell Dow

Keyword(s):

Xanthomonas Campestris ◽

Carbon Sources ◽

Regulatory System ◽

Extracellular Polysaccharide ◽

In Planta ◽

Glucuronidase Activity ◽

Gum Gene Cluster ◽

Diffusible Signal Factor ◽

Turnip Leaves

The gum gene cluster of Xanthomonas campestris pv. campestris comprises 12 genes whose products are involved in the biosynthesis of the extracellular polysaccharide xanthan. These genes are expressed primarily as an operon from a promoter upstream of the first gene, gumB. Although the regulation of xanthan synthesis in vitro has been well studied, nothing is known of its regulation in planta. A reporter plasmid was constructed in which the promoter region of the gum operon was fused to gusA. In liquid cultures, the expression of the gumgusA reporter was correlated closely with the production of xanthan, although a low basal level of β-glucuronidase activity was seen in the absence of added carbon sources when xanthan production was very low. The expression of the gumgusA fusion also was subject to positive regulation by rpfF, which is responsible for the synthesis of the diffusible signal factor (DSF). The expression of the gumgusA fusion in bacteria recovered from inoculated turnip leaves was maximal at the later phases of growth and was subject to regulation by rpfF. These results provide indirect support for the operation of the DSF regulatory system in bacteria in planta.

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Identification of the functions of 4-coumarate-CoA ligase/ acyl-CoA synthetase paralogs in potato

10.1101/2021.07.06.451337 ◽

2021 ◽

Author(s):

Matías Ariel Valiñas ◽

Arjen ten Have ◽

Adriana Balbina Andreu

Keyword(s):

3D Structure ◽

Class Ii ◽

Fatty Acyl ◽

Protein Family ◽

Class I ◽

Marker Genes ◽

Coa Ligase ◽

Coa Transferase ◽

Binding Cleft

Background: The 4CL/ ACS protein family is well known for its 4-coumarate-CoA ligase (4CL) enzymes but there are many aspects of this family that are still unclear or generally known. Cytosolic class I and class II 4CL enzymes control the biosynthesis of lignin/ suberin and flavonoids, respectively. Many 4CL homologs have broad substrate permissiveness in vitro and have no clear cut function. However, it has been demonstrated unequivocally that a peroxisomal 4CL-like homolog from Arabidopsis efficiently uses p-coumarate for ubiquinone biosynthesis. Another homolog has been shown to act as a fatty acyl-CoA synthetase and yet another as OPDA-CoA ligase. Hence, despite this knowledge, most homologs remain annotated as 4CL-like whereas other researches study the ACS protein family. Results: We set out identify the specific functions of 4CL/ ACS homologs, specifically in order to study the 4CL family in Solanum tuberosum. An in depth phylogenetic analysis was done. Using clustering techniques, functional annotation and taxonomic signals, three major clades were depicted. Clade 1 is composed of class I from monocotyledons, class I from dicotyledons and class II canonical 4CL enzymes subclades. Specificity determining positions and 3D structure analysis shows that clade 2 cytosolic 4CL-like enzymes show a rather different binding cleft and presumably use medium- to long-chain fatty acids. Clade 3 is composed of five subclades, four of which have a broad taxonomic contribution and a similar binding cleft as 4CLs whereas a fifth, specific for dicotyledons shows a significantly different binding pocket. The potato 4CL family comprises four class I (St4CL-I(A-D)) and one class II (St4CL-II) members. Transcript levels of St4CLs and of marker genes of the flavonoid (chalcone synthase, CHS) and suberin (feruloyl-CoA transferase, FHT) pathways were determined by qRT-PCR in flesh and skin from Andean varieties. St4CL-IA was barely detected in the skin of some varieties whereas St4CL-IB did not show a clear pattern. St4CL-IC and St4CL-ID could not be detected. St4CL-II expression pattern was similar to CHS. St4CL-IA and St4CL-IB were induced by wounding as did FHT whereas St4CL-II and CHS expression was repressed. Constitutive and wound-induced expression suggests that St4CL-IA and St4CL-IB isoforms are likely involved in soluble and/ or suberin-bound phenolic compounds while St4CL-II appears to be involved in flavonoid biosynthesis.

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NovelXanthomonas campestrisLong-Chain-Specific 3-Oxoacyl-Acyl Carrier Protein Reductase Involved in Diffusible Signal Factor Synthesis

mBio ◽

10.1128/mbio.00596-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 7

Author(s):

Zhe Hu ◽

Huijuan Dong ◽

Jin-Cheng Ma ◽

Yonghong Yu ◽

Kai-Hui Li ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Xanthomonas Campestris ◽

Octanoic Acid ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Carrier Protein ◽

Content Type ◽

Diffusible Signal Factor ◽

Acyl Chains

ABSTRACTThe precursors of the diffusible signal factor (DSF) family signals ofXanthomonas campestrispv.campestrisare 3-hydroxyacyl-acyl carrier protein (3-hydroxyacyl-ACP) thioesters having acyl chains of 12 to 13 carbon atoms produced by the fatty acid biosynthetic pathway. We report a novel 3-oxoacyl-ACP reductase encoded by theX. campestrispv.campestrisXCC0416 gene (fabG2), which is unable to participate in the initial steps of fatty acyl synthesis. This was shown by the failure of FabG2 expression to allow growth at the nonpermissive temperature of anEscherichia colifabGtemperature-sensitive strain. However, when transformed into theE. colistrain together with a plasmid bearing theVibrio harveyiacyl-ACP synthetase gene (aasS), growth proceeded, but only when the medium contained octanoic acid.In vitroassays showed that FabG2 catalyzes the reduction of long-chain (≥C8) 3-oxoacyl-ACPs to 3-hydroxyacyl-ACPs but is only weakly active with shorter-chain (C4, C6) substrates. FabG1, the housekeeping 3-oxoacyl-ACP reductase encoded within the fatty acid synthesis gene cluster, could be deleted in a strain that overexpressedfabG2but only in octanoic acid-supplemented media. Growth of theX. campestrispv.campestrisΔfabG1strain overexpressingfabG2requiredfabHfor growth with octanoic acid, indicating that octanoyl coenzyme A is elongated byX. campestrispv.campestrisfabH. Deletion offabG2reduced DSF family signal production, whereas overproduction of either FabG1 or FabG2 in the ΔfabG2strain restored DSF family signal levels.IMPORTANCEQuorum sensing mediated by DSF signaling molecules regulates pathogenesis in several different phytopathogenic bacteria, includingXanthomonas campestrispv.campestris. DSF signaling also plays a key role in infection by the human pathogenBurkholderia cepacia. The acyl chains of the DSF molecules are diverted and remodeled from a key intermediate of the fatty acid synthesis pathway. We report aXanthomonas campestrispv.campestrisfatty acid synthesis enzyme, FabG2, of novel specificity that seems tailored to provide DSF signaling molecule precursors.

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Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

The Journal of Cell Biology ◽

10.1083/jcb.201808133 ◽

2019 ◽

Vol 218 (4) ◽

pp. 1335-1351 ◽

Cited By ~ 38

Author(s):

Sanchari Datta ◽

Yang Liu ◽

Hanaa Hariri ◽

Jade Bowerman ◽

W. Mike Henne

Keyword(s):

Fatty Acids ◽

Endoplasmic Reticulum ◽

Cerebellar Ataxia ◽

Lipid Droplets ◽

Fatty Acyl ◽

Droplet Growth ◽

Sorting Nexin ◽

Coa Ligase ◽

In Trans ◽

Time Point

Lipid droplets (LDs) are nutrient reservoirs used by cells to maintain homeostasis. Nascent droplets form on the endoplasmic reticulum (ER) and grow following an influx of exogenous fatty acids (FAs). The budding of LDs requires extensive ER–LD crosstalk, but how this is regulated remains poorly understood. Here, we show that sorting nexin protein Snx14, an ER-resident protein associated with the cerebellar ataxia SCAR20, localizes to ER–LD contacts following FA treatment, where it promotes LD maturation. Using proximity-based APEX technology and topological dissection, we show that Snx14 accumulates specifically at ER–LD contacts independently of Seipin, where it remains ER-anchored and binds LDs in trans. SNX14KO cells exhibit perturbed LD morphology, whereas Snx14 overexpression promotes LD biogenesis and extends ER–LD contacts. Multi–time point imaging reveals that Snx14 is recruited to ER microdomains containing the fatty acyl-CoA ligase ACSL3, where nascent LDs bud. We propose that Snx14 is a novel marker for ER–LD contacts and regulates FA-stimulated LD growth.

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