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An important regulatory role for ethylene-responsive transcription factors (ERFs) is in plant growth and development, stress response, and hormone signaling. However, AP2/ERF family genes in mango have not been systematically studied. In this study, a total of 113 AP2/ERF family genes were identified from the mango genome and phylogenetically classified into five subfamilies: AP2 (28 genes), DREB (42 genes), ERF (33 genes), RAV (6 genes), and Soloist (4 genes). Of these, the ERF family, in conjunction with Arabidopsis and rice, forms a phylogenetic tree divided into seven groups, five of which have MiERF members. Analysis of gene structure and cis-elements showed that each MiERF gene contains only one AP2 structural domain, and that MiERF genes contain a large number of cis-elements associated with hormone signaling and stress response. Collinearity tests revealed a high degree of homology between MiERFs and CsERFs. Tissue-specific and stress-responsive expression profiling revealed that MiERF genes are primarily involved in the regulation of reproductive growth and are differentially and positively expressed in response to external hormones and pathogenic bacteria. Physiological results from a gain-of-function analysis of MiERF4 transiently overexpressed in tobacco and mango showed that transient expression of MiERF4 resulted in decreased colony count and callose deposition, as well as varying degrees of response to hormonal signals such as ETH, JA, and SA. Thus, MiERF4 may be involved in the JA/ETH signaling pathway to enhance plant defense against pathogenic bacteria. This study provides a basis for further research on the function and regulation of MiERF genes and lays a foundation for the selection of disease-resistant genes in mango., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

High-temperature-requirement protein A (HtrA) family proteins play important roles in controlling protein quality and are recognized as virulence factors in numerous animal and human bacterial pathogens. The role of HtrA family proteins in plant pathogens remains largely unexplored. Here, we investigated the HtrA family protein, DegQ, in the crucifer black rot pathogen Xanthomonas campestris pathovar campestris (Xcc). DegQ is essential for bacterial attachment and full virulence of Xcc. Moreover, the degQ mutant strain showed increased sensitivity to heat treatment and sodium dodecyl sulfate. Expressing the intact degQ gene in trans in the degQ mutant could reverse the observed phenotypic changes. In addition, we demonstrated that the DegQ protein exhibited chaperone-like activity. Transcriptional analysis displayed that degQ expression was induced under heat treatment. Our results contribute to understanding the function and expression of DegQ of Xcc for the first time and provide a novel perspective about HtrA family proteins in plant pathogen., (© 2024 Wiley‐VCH GmbH.)

Background: Copper-induced gene expression in Xanthomonas campestris pv. campestris (Xcc) is typically evaluated using targeted approaches involving qPCR. The global response to copper stress in Xcc and resistance to metal induced damage is not well understood. However, homologs of heavy metal efflux genes from the related Stenotrophomonas genus are found in Xanthomonas which suggests that metal related efflux may also be present., Methods and Results: Gene expression in Xcc strain BrA1 exposed to 0.8 mM CuSO 4 .5H 2 O for 15 minutes was captured using RNA-seq analysis. Changes in expression was noted for genes related to general stress responses and oxidoreductases, biofilm formation, protein folding chaperones, heat-shock proteins, membrane lipid profile, multiple drug and efflux (MDR) transporters, and DNA repair were documented. At this timepoint only the cohL (copper homeostasis/tolerance) gene was upregulated as well as a chromosomal czcCBA efflux operon. An additional screen up to 4 hrs using qPCR was conducted using a wider range of heavy metals. Target genes included a cop-containing heavy metal resistance island and putative metal efflux genes. Several efflux pumps, including a copper resistance associated homolog from S. maltophilia, were upregulated under toxic copper stress. However, these pumps were also upregulated in response to other toxic heavy metals. Additionally, the temporal expression of the coh and cop operons was also observed, demonstrating co-expression of tolerance responses and later activation of part of the cop operon., Conclusions: Overall, initial transcriptional responses focused on combating oxidative stress, mitigating protein damage and potentially increasing resistance to heavy metals and other biocides. A putative copper responsive efflux gene and others which might play a role in broader heavy metal resistance were also identified. Furthermore, the expression patterns of the cop operon in conjunction with other copper responsive genes allowed for a better understanding of the fate of copper ions in Xanthomonas. This work provides useful evidence for further evaluating MDR and other efflux pumps in metal-specific homeostasis and tolerance phenotypes in the Xanthomonas genus. Furthermore, non-canonical copper tolerance and resistance efflux pumps were potentially identified. These findings have implications for interpreting MIC differences among strains with homologous copLAB resistance genes, understanding survival under copper stress, and resistance in disease management., (© 2024. The Author(s).)

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in the biosynthesis of nicotinamide adenine dinucleotide (NAD + ) via the nicotinamide (NAM) salvage pathway. While the structural biochemistry of eukaryote NAMPT has been well studied, the catalysis mechanism of prokaryote NAMPT at the molecular level remains largely unclear. Here, we demonstrated the NAMPT-mediated salvage pathway is functional in the Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) for the synthesis of NAD + , and the enzyme activity of NAMPT in this bacterium is significantly higher than that of human NAMPT in vitro. Our structural analyses of Xcc NAMPT, both in isolation and in complex with either the substrate NAM or the product nicotinamide mononucleotide (NMN), uncovered significant details of substrate recognition. Specifically, we revealed the presence of a NAM binding tunnel that connects the active site, and this tunnel is essential for both catalysis and inhibitor binding. We further demonstrated that NAM binding in the tunnel has a positive cooperative effect with NAM binding in the catalytic site. Additionally, we discovered that phosphorylation of the His residue at position 229 enhances the substrate binding affinity of Xcc NAMPT and is important for its catalytic activity. This work reveals the importance of NAMPT in bacterial NAD + synthesis and provides insights into the substrate recognition and the catalytic mechanism of bacterial type II phosphoribosyltransferases., (© 2024. The Author(s).)

Xanthan gum, an anionic polysaccharide with an exceptionally high molecular weight, is produced by the bacterium Xanthomonas sp. It is a versatile compound that has been utilized in various industries for decades. Xanthan gum was the second exopolysaccharide to be commercially produced, following dextran. In 1969, the US Food and Drug Administration (FDA) approved xanthan gum for use in the food and pharmaceutical industries. The food industry values xanthan gum for its exceptional rheological properties, which make it a popular thickening agent in many products. Meanwhile, the cosmetics industry capitalizes on xanthan gum's ability to form stable emulsions. The industrial production process of xanthan gum involves fermenting Xanthomonas in a medium that contains glucose, sucrose, starch, etc. as a substrate and other necessary nutrients to facilitate growth. This is achieved through batch fermentation under optimal conditions. However, the increasing costs of glucose in recent years have made the production of xanthan economically unviable. Therefore, many researchers have investigated alternative, cost-effective substrates for xanthan production, using various modified and unmodified raw materials. The objective of this analysis is to investigate how utilizing different raw materials can improve the cost-efficient production of xanthan gum., (© 2023 American Institute of Chemical Engineers.)

Background: Cruciferous black rot is caused by Xanthomonas campestris pv. campestris (Xcc) infection and is a widespread disease worldwide. Excessive and repeated use of bactericide is an important cause of the development of bacterial resistance. It is imperative to take new approaches to screening compounds that target virulence factors rather than kill bacterial pathogens. The type III secretion system (T3SS) invades a variety of cells by transporting virulence effector factors into the cytoplasm and is an attractive antitoxic target. Toward the search of new T3SS inhibitors, an alternative series of novel pyrimidin-4-one derivatives were designed and synthesized and assessed for their effect in blocking the virulence., Results: All of the target compounds were characterized by proton ( 1 H) nuclear magnetic resonance (NMR), carbon-13 ( 13 C) NMR, fluorine-19 ( 19 F) NMR and high-resolution mass spectrometry (HRMS). All compounds were evaluated using high-throughput screening systems against Xcc. The results of the biological activity test revealed that the compound SPF-9 could highly inhibit the activity of xopN gene promoter and the hypersensitivity (HR) of tobacco without affecting bacterial growth. Moreover, messenger RNA (mRNA) level measurements showed that compound SPF-9 inhibited the expression of some representative genes (hrp/hrc genes). Compound SPF-9 weakened the pathogenicity of Xcc to Raphanus sativus L., Conclusion: Compound SPF-9 has good potential for further development as a novel T3SS inhibitor against Xcc. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Ribonucleases (RNases) play critical roles in RNA metabolism and are collectively essential for cell viability. However, most knowledge about bacterial RNases comes from the studies on Escherichia coli ; very little is known about the RNases in plant pathogens. The crucifer black rot pathogen Xanthomonas campestris pv. campestris ( Xcc ) encodes 15 RNases, but none of them has been functionally characterized. Here, we report the physiological function of the exoribonuclease RNase D in Xcc and provide evidence demonstrating that the Xcc RNase D is involved in 5S rRNA degradation and exopolysaccharide (EPS) production. Our work shows that the growth and virulence of Xcc were not affected by deletion of RNase D but were severely attenuated by RNase D overexpression. However, deletion of RNase D in Xcc resulted in a significant reduction in EPS production. In addition, either deletion or overexpression of RNase D in Xcc did not influence the tRNAs tested, inconsistent with the finding in E. coli that the primary function of RNase D is to participate in tRNA maturation and its overexpression degrades tRNAs. More importantly, deletion, overexpression, and in vitro enzymatic analyses revealed that the Xcc RNase D degrades 5S rRNA but not 16S and 23S rRNAs that share an operon with 5S rRNA. Our results suggest that Xcc employs RNase D to realize specific modulation of the cellular 5S rRNA content after transcription and maturation whenever necessary. The finding expands our knowledge about the function of RNase D in bacteria., Competing Interests: The author(s) declare no conflict of interest.

Exo70B1 is a protein subunit of the exocyst complex with a crucial role in a variety of cell mechanisms, including immune responses against pathogens. The calcium-dependent kinase 5 (CPK5) of Arabidopsis thaliana (hereafter Arabidopsis), phosphorylates AtExo70B1 upon functional disruption. We previously reported that, the Xanthomonas campestris pv. campestris effector XopP compromises AtExo70B1, while bypassing the host's hypersensitive response, in a way that is still unclear. Herein we designed an experimental approach, which includes biophysical, biochemical, and molecular assays and is based on structural and functional predictions, utilizing AplhaFold and DALI online servers, respectively, in order to characterize the in vivo XccXopP function. The interaction between AtExo70B1 and XccXopP was found very stable in high temperatures, while AtExo70B1 appeared to be phosphorylated at XccXopP-expressing transgenic Arabidopsis. XccXopP revealed similarities with known mammalian kinases and phosphorylated AtExo70B1 at Ser107, Ser111, Ser248, Thr309, and Thr364. Moreover, XccXopP protected AtExo70B1 from AtCPK5 phosphorylation. Together these findings show that XccXopP is an effector, which not only functions as a novel serine/threonine kinase upon its host target AtExo70B1 but also protects the latter from the innate AtCPK5 phosphorylation, in order to bypass the host's immune responses. Data are available via ProteomeXchange with the identifier PXD041405., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Pathogenic Xanthomonas bacteria cause disease on more than 400 plant species. These Gram-negative bacteria utilize the type III secretion system to inject type III effector proteins (T3Es) directly into the plant cell cytosol where they can manipulate plant pathways to promote virulence. The host range of a given Xanthomonas species is limited, and T3E repertoires are specialized during interactions with specific plant species. Some effectors, however, are retained across most strains, such as Xanthomonas Outer Protein L (XopL). As an 'ancestral' effector, XopL contributes to the virulence of multiple xanthomonads, infecting diverse plant species. XopL homologs harbor a combination of a leucine-rich-repeat (LRR) domain and an XL-box which has E3 ligase activity. Despite similar domain structure there is evidence to suggest that XopL function has diverged, exemplified by the finding that XopLs expressed in plants often display bacterial species-dependent differences in their sub-cellular localization and plant cell death reactions. We found that XopL from X. euvesicatoria (XopLXe) directly associates with plant microtubules (MTs) and causes strong cell death in agroinfection assays in N. benthamiana. Localization of XopLXe homologs from three additional Xanthomonas species, of diverse infection strategy and plant host, revealed that the distantly related X. campestris pv. campestris harbors a XopL (XopLXcc) that fails to localize to MTs and to cause plant cell death. Comparative sequence analyses of MT-binding XopLs and XopLXcc identified a proline-rich-region (PRR)/α-helical region important for MT localization. Functional analyses of XopLXe truncations and amino acid exchanges within the PRR suggest that MT-localized XopL activity is required for plant cell death reactions. This study exemplifies how the study of a T3E within the context of a genus rather than a single species can shed light on how effector localization is linked to biochemical activity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ortmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Many Gram-negative bacteria use quorum sensing (QS) signal molecules to monitor their local population density and to coordinate their collective behaviors. The diffusible signal factor (DSF) family represents an intriguing type of QS signal to mediate intraspecies and interspecies communication. Recently, accumulating evidence demonstrates the role of DSF in mediating inter-kingdom communication between DSF-producing bacteria and plants. However, the regulatory mechanism of DSF during the Xanthomonas -plant interactions remain unclear., Methods: Plants were pretreated with different concentration of DSF and subsequent inoculated with pathogen Xanthomonas campestris pv. campestris (Xcc) . Pathogenicity, phynotypic analysis, transcriptome combined with metabolome analysis, genetic analysis and gene expression analysis were used to evaluate the priming effects of DSF on plant disease resistance., Results: We found that the low concentration of DSF could prime plant immunity against Xcc in both Brassica oleracea and Arabidopsis thaliana . Pretreatment with DSF and subsequent pathogen invasion triggered an augmented burst of ROS by DCFH-DA and DAB staining. CAT application could attenuate the level of ROS induced by DSF. The expression of RBOHD and RBOHF were up-regulated and the activities of antioxidases POD increased after DSF treatment followed by Xcc inoculation. Transcriptome combined with metabolome analysis showed that plant hormone jasmonic acid (JA) signaling involved in DSF-primed resistance to Xcc in Arabidopsis. The expression of JA synthesis genes ( AOC2, AOS, LOX2, OPR3 and JAR1 ), transportor gene ( JAT1 ), regulator genes ( JAZ1 and MYC2 ) and responsive genes ( VSP2, PDF1.2 and Thi2.1 ) were up-regulated significantly by DSF upon Xcc challenge. The primed effects were not observed in JA relevant mutant coi1-1 and jar1-1 ., Conclusion: These results indicated that DSF-primed resistance against Xcc was dependent on the JA pathway. Our findings advanced the understanding of QS signal-mediated communication and provide a new strategy for the control of black rot in Brassica oleracea ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhao, Liu, Song, Zhai, He, Ma, Zhao, Jia and Song.)

Employing aerobic fermentation, Gram-negative bacteria belonging to the genus Xanthomonas produce the high molecular weight natural heteropolysaccharide known as xanthan. It has various amounts of O-acetyl and pyruvyl residues together with D-glucosyl, D-mannosyl, and D-glucuronyl acid residues in a molar ratio of 2:2:1. The unique structure of xanthan allowed its various applications in a wide range of industries such as the food industry, pharmacology, cosmetics and enhanced oil recovery primarily in petroleum. The cultivation medium used in the manufacture of this biopolymer is critical. Many attempts have been undertaken to generate xanthan gum from agro-based and food industry wastes since producing xanthan gum from synthetic media is expensive. Optimal composition and processing parameters must also be considered to achieve an economically viable manufacturing process. There have been several attempts to adjust the nutrient content and feeding method, temperature, pH, agitation and the use of antifoam in xanthan fermentations. Various modifications in technological approaches have been applied to enhance its physicochemical properties which showed significant improvement in the area studied. This review describes the biosynthesis production of xanthan with an emphasis on the importance of the upstream processes involving medium, processing parameters, and other factors that significantly contributed to the final application of this precious polysaccharide., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Most bacteria use type II fatty acid synthesis (FAS) systems for synthesizing fatty acids, of which the conserved FabA-FabB pathway is considered to be crucial for unsaturated fatty acid (UFA) synthesis in gram-negative bacteria. Xanthomonas campestris pv. campestris , the phytopathogen of black rot disease in crucifers, produces higher quantities of UFAs under low-temperature conditions for increasing membrane fluidity. The fabA and fabB genes were identified in the X. campestris pv. campestris genome by BLAST analysis; however, the growth of the X. campestris pv. campestris fabA and fabB deletion mutants was comparable to that of the wild-type strain in nutrient and minimal media. The X. campestris pv. campestris Δ fabA and Δ fabB strains produced large quantities of UFAs and, altogether, these results indicated that the FabA-FabB pathway is not essential for growth or UFA synthesis in X. campestris pv. campestris . We also observed that the expression of X. campestris pv. campestris fabA and fabB restored the growth of the temperature-sensitive Escherichia coli fabA and fabB mutants CL104 and CY242, respectively, under non-permissive conditions. The in-vitro assays demonstrated that the FabA and FabB proteins of X. campestris pv. campestris catalyzed FAS. Our study also demonstrated that the production of diffusible signal factor family signals that mediate quorum sensing was higher in the X. campestris pv. campestris Δ fabA and Δ fabB strains and greatly reduced in the complementary strains, which exhibited reduced swimming motility and attenuated host-plant pathogenicity. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Plant secondary metabolites perform numerous functions in the interactions between plants and pathogens. However, little is known about the precise mechanisms underlying their contribution to the direct inhibition of pathogen growth and virulence in planta. Here, we show that the secondary metabolite sulforaphane (SFN) in crucifers inhibits the growth, virulence, and ability of Xanthomonas species to adapt to oxidative stress, which is essential for the successful infection of host plants by phytopathogens. The transcription of oxidative stress detoxification-related genes (catalase [katA and katG] and alkylhydroperoxide-NADPH oxidoreductase subunit C [ahpC]) was substantially inhibited by SFN in Xanthomonas campestris pv. campestris (Xcc), and this phenomenon was most obvious in sax gene mutants sensitive to SFN. By performing microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA), we observed that SFN directly bound to the virulence-related redox-sensing transcription factor OxyR and weakened the ability of OxyR to bind to the promoters of oxidative stress detoxification-related genes. Collectively, these results illustrate that SFN directly targets OxyR to inhibit the bacterial adaptation to oxidative stress, thereby decreasing bacterial virulence. Interestingly, this phenomenon occurs in multiple Xanthomonas species. This study provides novel insights into the molecular mechanisms by which SFN limits Xanthomonas adaptation to oxidative stress and virulence, and the findings will facilitate future studies on the use of SFN as a biopesticide to control Xanthomonas., (© 2022 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The α-glucosidase (EC 3.2.1.20) XgtA produced by Xanthomonas campestris shows high α-glucosyl transfer activity toward alcoholic and phenolic hydroxyl groups. Ethyl vanillin-α-glucoside, a precursor-aroma compound with improved water solubility and thermal stability, can be synthesized through the transglycosylation of ethyl vanillin by XgtA. However, its low ethyl vanillin-α-glucoside yield and ability to hydrolyze ethyl vanillin-α-glucoside limits for industrial applications. Rational design and site-directed mutagenesis were employed to generate three variants of X. campestris α-glucosidase, L145I, S272T, and L145I/S272T, with improved transglycosylation activity toward EV. The highest yield is up to 52.41% by L145I/S272T, which also displayed remarkably lower hydrolysis activity toward the glycoside product EVG compared to XgtA. These results also showed that the mutation in sugar-binding subsite + 1 is more effective than subsite -1 for enhancing the ratio of transglycosylation/hydrolysis for the α-glucosidase XgtA., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in numerous regulatory cell processes. Xanthomonas campestris pv. campestris is a Gram-negative pathogen that can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris has several clp genes, namely, clpS, clpA, clpX, clpP, clpQ, and clpY. Among these genes, only clpX and clpP is known to be required for pathogenicity. Here, we focused on two uncharacterized clp genes (clpS and clpA) that encode the adaptor (ClpS) and ATPase subunit (ClpA) of the ClpAP protease complex. Transcriptional analysis revealed that the expression of clpS and clpA was growth phase-dependent and affected by the growth temperature. The inactivation of clpA, but not of clpS, resulted in susceptibility to high temperature and attenuated virulence in the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 were the amino acid residues critical for ClpA function in heat tolerance. The protein expression profile shown by the clpA mutant in response to heat stress was different from that exhibited by the wild type. In summary, we characterized two clp genes (clpS and clpA) by examining their expression profiles and functions in different processes, including stress tolerance and pathogenicity. We demonstrated that clpS and clpA were expressed in a temperature-dependent manner and that clpA was required for the survival at high temperature and full virulence of X. campestris pv. campestris. This work represents the first time that clpS and clpA were characterized in Xanthomonas., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

As a critical part of plant immunity, cells that are attacked by pathogens undergo rapid transcriptional reprogramming to minimize virulence. Many bacterial phytopathogens use type III effector (T3E) proteins to interfere with plant defense responses, including this transcriptional reprogramming. Here, we show that Xanthomonas outer protein S (XopS), a T3E of Xanthomonas campestris pv. vesicatoria (Xcv), interacts with and inhibits proteasomal degradation of WRKY40, a transcriptional regulator of defense gene expression. Virus-induced gene silencing of WRKY40 in pepper (Capsicum annuum) enhanced plant tolerance to Xcv infection, indicating that WRKY40 represses immunity. Stabilization of WRKY40 by XopS reduces the expression of its targets, which include salicylic acid-responsive genes and the jasmonic acid signaling repressor JAZ8. Xcv bacteria lacking XopS display significantly reduced virulence when surface inoculated onto susceptible pepper leaves. XopS delivery by Xcv, as well as ectopic expression of XopS in Arabidopsis thaliana or Nicotiana benthamiana, prevented stomatal closure in response to bacteria and biotic elicitors. Silencing WRKY40 in pepper or N. benthamiana abolished XopS's ability to prevent stomatal closure. This suggests that XopS interferes with both preinvasion and apoplastic defense by manipulating WRKY40 stability and downstream gene expression, eventually altering phytohormone crosstalk to promote pathogen proliferation., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

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, and it is the predominant in planta quorum-sensing signal in Xanthomonas campestris. 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 does not seem to be 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 activity to degrade the bacterial second messenger cyclic di-GMP. We demonstrated that BDSF is prone to degradation by RpfB, a critical fatty acyl coenzyme A 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. IMPORTANCE The diffusible signal factor (DSF) family consists of quorum-sensing signals employed by Gram-negative bacteria. These signals are a group of cis -2-unsaturated fatty acids, such as DSF, BDSF, IDSF, CDSF, and SDSF. However, the functional divergence of various DSF signals remains unclear. The present study demonstrates that though BDSF is a low active quorum-sensing signal, it binds histidine kinase RpfC with a higher affinity and activates RpfC autophosphorylation to the similar level as DSF. Rather than regulation of enzymatic activities of RpfC and its cognate response regulator RpfG encoding a c-di-GMP hydrolase, BDSF is prone to degradation in bacterial cells by RpfB, which effectively avoided the inhibition of bacterial growth by accumulating high concentrations of BDSF. Therefore, our study sheds new light on the functional differences of quorum-sensing signals and shows that bacteria balance quorum sensing and growth by fine-tuning concentrations of signaling chemicals.

In Xanthomonas spp., the biosynthesis of the yellow pigment xanthomonadin and fatty acids originates in the type II polyketide synthase (PKS II) and fatty acid synthase (FAS) pathways, respectively. The acyl carrier protein (ACP) is the central component of PKS II and FAS and requires posttranslational phosphopantetheinylation to initiate these pathways. In this study, for the first time, we demonstrate that the posttranslational modification of ACPs in X. campestris pv. campestris is performed by an essential 4'-phosphopantetheinyl transferase (PPTase), XcHetI (encoded by Xc_4132). X. campestris pv. campestris strain XchetI could not be deleted from the X. campestris pv. campestris genome unless another PPTase-encoding gene such as Escherichia coli acpS or Pseudomonas aeruginosa pcpS was present. Compared with wild-type strain X. campestris pv. campestris 8004 and mutant XchetI :: PapcpS , strain XchetI :: EcacpS failed to generate xanthomonadin pigments and displayed reduced pathogenicity for the host plant, Brassica oleracea . Further experiments showed that the expression of XchetI restored the growth of E. coli acpS mutant HT253 and, when a plasmid bearing XchetI was introduced into P. aeruginosa , pcpS , which encodes the sole PPTase in P. aeruginosa , could be deleted. In in vitro enzymatic assays, XcHetI catalyzed the transformation of 4'-phosphopantetheine from coenzyme A to two X. campestris pv. campestris apo -acyl carrier proteins, XcAcpP and XcAcpC. All of these findings indicate that XcHetI is a surfactin PPTase-like PPTase with a broad substrate preference. Moreover, the HetI-like PPTase is ubiquitously conserved in Xanthomonas spp., making it a potential new drug target for the prevention of plant diseases caused by Xanthomonas .[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Background: Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium that can cause black rot disease in crucifers. The lipoprotein outer membrane localization (Lol) system is involved in the lipoprotein sorting to the outer membrane. Although Xcc has a set of annotated lol genes, there is still little known about the physiological role in this phytopathogen. In this study, we aimed to characterize the role of LolB of Xcc in bacterial attachment, stress tolerance, and virulence., Results: To characterize the role of LolB, lolB mutant was constructed and phenotypic evaluation was performed. The lolB mutant revealed reductions in bacterial attachment, extracellular enzyme production, and virulence. Mutation of lolB also resulted in reduced tolerance to a myriad of stresses, including heat and a range of membrane-perturbing agents. Trans-complementation of lolB mutant with intact lolB gene reverted these altered phenotypes to the wild-type levels. From subsequent reporter assay and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, the expression of genes that encode the major extracellular enzymes and the stress-related proteins was reduced after lolB mutation., Conclusions: The results in this work contribute to the functional understanding of lolB in Xanthomonas for the first time, and provide new insights into the function of lolB in bacteria., (© 2022. The Author(s).)

Oil recovery is a challenge and microbial enhanced oil recovery is an option. We theorized that the use of produced water (PW) with photo-stimulation could influence both production and viscosity of Xanthan gum. This study aimed at the evaluation of the effect of photo-stimulation by λ630 ± 1 ηm LED light on the biosynthesis of Xanthan gum produced by Xanthomonas campestris IBSBF 2103 strain reusing PW of the oil industry. We assessed the effect of photo-stimulation by LED light (λ630 nm) on the biosynthesis of Xanthan gum produced by X. campestris in medium containing produced water. Different energy densities applied during the microbial growth phase were tested. The highest production was achieved when using 12 J/cm 2 LED light (p < 0.01). Three protocols were assessed: Non-irradiated (Control), Irradiation with LED light during the growth phase (LED growth ) and Irradiation with LED light during both growth and production phases (LED growth+production ). Both the amount and viscosity of the xanthan gum was significantly higher (p < 0.01) in the group LED growth+production . The study showed that LED irradiation (λ630 ± 1 ηm) during both the growth and production phases of the biopolymer increased both the production and viscosity of Xanthan gum., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bacteria harbour several abundant small DNA-binding proteins known as nucleoid-associated proteins (NAPs) that contribute to the structure of the bacterial nucleoid as well as to gene regulation. Although the function of NAPs as global transcriptional regulators has been comprehensively studied in the model organism Escherichia coli, their regulatory functions in other bacteria remain relatively poorly understood. Xanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium that causes black rot disease in almost all members of the crucifer family. In previous work, we demonstrated that a Fis homologue protein, which we named Fis-like protein (Flp), contributes to the regulation of virulence, type III secretion, and a series of other phenotypes in Xcc. Here we have examined the role of XC&#95;1355, which is predicted to encode a DNA-binding protein belonging to the HU family herein named HU-like protein (Hlp). We show that mutation of XC&#95;1355 in Xcc reduces the virulence, extracellular polysaccharide production, and cell motility, but has no effect on the production of extracellular enzymes and induction of the hypersensitive response. These data together with transcriptome analysis indicate that hlp is a previously uncharacterized gene involved in virulence that has partially overlapping and complementary functions with flp in Xcc, although the two regulators have opposite effects on the expression of genes involved in type III secretion. The findings add to our understanding of the complex regulatory pathways that act to regulate virulence in Xcc., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that causes black rot in host. It is an important model strain for studying the interaction between the phytopathogen and plants. In Xcc, global transcription regulator HpaR1 that belongs to the GntR family regulates many cellular processes such as the movement and synthesis of extracellular polysaccharides and extracellular enzymes, and is associated with hypersensitive response (HR) and pathogenicity. On the other hand, the global transcriptional regulator Clp regulates the secretion and synthesis of extracellular enzymes and extracellular polysaccharides, and is associated with the pathogenicity of Xanthomonas. Previous studies have shown that both HpaR1 and Clp bind to the promoter region of the glycoside hydrolase encoding gene (named ghy gene). This study investigates the molecular mechanism of the co-regulation of HpaR1 and Clp on the expression of ghy gene. Through electrophoresis mobility shift assay (EMSA), we found that both HpaR1 and Clp bind to the promoter regions of gene ghy in vitro. Both HpaR1 and Clp also bind to the promoter regions of gene ghy in vivo by chromatin immunoprecipitation (ChIP) assays. DNase I footprinting and 5'-RACE assays showed that both HpaR1 and Clp bind to the -35 region upstream of the ghy promoter. The HpaR1 binding site was located upstream of the Clp binding site. RT-qPCR and in vitro transcription assays showed that HpaR1 negatively while Clp positively regulates the transcription of gene ghy. Furthermore, HpaR1 inhibits the activation of Clp on the transcription of gene ghy in vitro. Our findings indicate that HpaR1 and Clp exhibit opposite effect on the transcription of gene ghy. It is speculated that HpaR1 may regulate the expression of gene ghy by inhibiting the activity of RNA polymerase.

Xanthan gum (XG), a bacterial polysaccharide has numerous valuable characteristics in the food, biomedical, pharmaceuticals, and agriculture sector. However, XG has also its particular limitations such as its vulnerability to microbial contamination, inadequate mechanical and thermal stability, unusable viscosity, and poor water solubility. Therefore, XG's structure and conformation need to be modified enzymatically, chemically, or physically to improve its optimistic features and decrease the formation of crystals, increase antioxidant ability, and radical scavenging activity. We have found out different means to modify XG and elaborate the importance and significance of the modified structure of XG. In this review, different enzymes are reviewed for XG degradation, which modifies their structure from different points (main chain or side chain). This article also reviews various physical methods (ultrasound, shear, pressure, sonication, annealing, and heat treatments) based on prevailing publications to alter XG conformation and produce low molecular weight (LMW) and less viscous end-product. Moreover, some chemical means are also discussed that result in modified XG through crosslinking, grafting, acetylation, pyruvation, as well as by applying different chemical agents. Overall, the current progress on XG degradation is very auspicious to develop a new molecule with considerable uses, in various industries with future assessments., (Copyright © 2021. Published by Elsevier B.V.)

As with many phytopathogenic bacteria, the virulence of Xanthomonas campestris pv. campestris , the causal agent of black rot disease in cruciferous plants, relies on secretion of a suite of extracellular enzymes that includes cellulase (endoglucanase), pectinase, protease, and amylase. Although the role in virulence of a number of these enzymes has been assessed, the contribution of amylase to X. campestris pv. campestris virulence has yet to be established. In this work, we investigated both the role of extracellular amylase in X. campestris pv. campestris virulence and the control of its expression. Deletion of XC3487 (here renamed amyA Xcc ), a putative amylase-encoding gene from the genome of X. campestris pv. campestris strain 8004, resulted in a complete loss of extracellular amylase activity and significant reduction in virulence. The extracellular amylase activity and virulence of the amyA Xcc mutant could be restored to the wild-type level by expressing amyA Xcc in trans. These results demonstrated that amyA Xcc is responsible for the extracellular amylase activity of X. campestris pv. campestris and indicated that extracellular amylase plays an important role in X. campestris pv. campestris virulence. We also found that the expression of amyA Xcc is strongly induced by starch and requires activation by the global posttranscriptional regulator RsmA. RsmA binds specifically to the 5'-untranslated region of amyA Xcc transcripts, suggesting that RsmA regulates amyA Xcc directly at the posttranscriptional level. Unexpectedly, in addition to posttranscriptional regulation, the use of a transcriptional reporter demonstrated that RsmA also regulates amyA Xcc expression at the transcriptional level, possibly by an indirect mechanism.

The intrinsically disordered region (IDR) is a preserved signature of phytobacterial type III effectors (T3Es). The T3E IDR is thought to mediate unfolding during translocation into the host cell and to avoid host defense by sequence diversification. Here, we demonstrate a mechanism of host subversion via the T3E IDR. We report that the Xanthomonas campestris T3E XopR undergoes liquid-liquid phase separation (LLPS) via multivalent IDR-mediated interactions that hijack the Arabidopsis actin cytoskeleton. XopR is gradually translocated into host cells during infection and forms a macromolecular complex with actin-binding proteins at the cell cortex. By tuning the physical-chemical properties of XopR-complex coacervates, XopR progressively manipulates multiple steps of actin assembly, including formin-mediated nucleation, crosslinking of F-actin, and actin depolymerization, which occurs through competition for actin-depolymerizing factor and depends on constituent stoichiometry. Our findings unravel a sophisticated strategy in which bacterial T3E subverts the host actin cytoskeleton via protein complex coacervation.

The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper and tomato plants. Pathogenicity of X. campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates bacterial effector proteins into plant cells. At least nine membrane-associated and cytoplasmic components of the secretion apparatus are homologous to corresponding Sct (secretion and cellular translocation) proteins from animal pathogens, suggesting a similar structural organisation of T3S systems in different bacterial species. T3S in X. campestris pv. vesicatoria also depends on non-conserved proteins with yet unknown function including the essential pathogenicity factor HrpB4. Here, we show that HrpB4 localises to the cytoplasm and the bacterial membranes and interacts with the cytoplasmic domain of the inner membrane (IM) ring component HrcD and the cytoplasmic HrcQ protein. The analysis of HrpB4 deletion derivatives revealed that deletion of the N- or C-terminal protein region affects the interaction of HrpB4 with HrcQ and HrcD as well as its contribution to pathogenicity. HrcQ is a component of the predicted sorting platform, which was identified in animal pathogens as a dynamic heterooligomeric protein complex and associates with the IM ring via SctK proteins. HrcQ complex formation was previously shown by fluorescent microscopy analysis and depends on the presence of the T3S system. In the present study, we provide experimental evidence that the absence of HrpB4 severely affects the docking of HrcQ complexes to the T3S system but does not significantly interfere with HrcQ complex formation in the bacterial cytoplasm. Taken together, our data suggest that HrpB4 links the predicted cytoplasmic sorting platform to the IM rings of the T3S system., (© 2021 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

The plant pathogen Xanthomonas is commonly found in biocontaminated bioreactors; however, few studies have evaluated the growth and impacts of this microorganism on bioreactors. In this study, we examined the characteristics of Xanthomonas campestris pv. campestris (Xcc). Our results showed that Xcc could reduce metal Fe (III) and decolorise methyl orange in vitro. Moreover, I-t and cyclic voltammetry curves showed that Xcc could generate bioelectricity and had two extracellular electron transfer pathways, similar to that of Shewanella. Based on the spectral analysis of intact cells and scanning electron microscopy analysis, one pathway was speculated to involve cytochrome C by direct contact with the pili or cell surface. The other pathway may involve indirect mediators, such as redox substrates, among extracellular polymeric substances. For the direct extracellular electron transfer process, the charge transfer coefficient α, electron number n, and the electron transfer rate constant k s were determined to be 0.49, 2.6, and 2.2 × 10 -3  s -1 , respectively. In the indirect extracellular electron transfer processes, the values of α, n, and k s were 0.52, 4, and 1.21 s -1 , respectively. Of these two transfer methods, indirect electron transfer is dominant and faster than direct electron transfer. Moreover, after mutation of the dsbD gene, which is important for indirect electron transfer, the electrochemical parameters α, n, and k s decreased. Our findings reveal a new anaerobic mechanism mediating the survival of Xcc during wastewater treatment, and may help develop new strategies for preventing Xcc growth during wastewater treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Plants respond to bacterial infection acutely with actin remodeling during plant immune responses. The mechanisms by which bacterial virulence factors (VFs) modulate plant actin polymerization remain enigmatic. Here, we show that plant-type-I formin serves as the molecular sensor for actin remodeling in response to two bacterial VFs: Xanthomonas campestris pv. campestris (Xcc) diffusible signal factor (DSF), and pathogen-associated molecular pattern (PAMP) flagellin in pattern-triggered immunity (PTI). Both VFs regulate actin assembly by tuning the clustering and nucleation activity of formin on the plasma membrane (PM) at the nano-sized scale. By being integrated within the cell-wall-PM-actin cytoskeleton (CW-PM-AC) continuum, the dynamic behavior and function of formins are highly dependent on each scaffold layer's composition within the CW-PM-AC continuum during both DSF and PTI signaling. Our results reveal a central mechanism for rapid actin remodeling during plant-bacteria interactions, in which bacterial signaling molecules fine tune plant formin nanoclustering in a host mechanical-structure-dependent manner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Semi-continuous production of xanthan gum using self-immobilized Xanthomonas campestris cells in biofilm reactors was studied. Fermentation was carried out using two different designs of biofilm reactor equipped with a) stainless-steel support (SSS) and b) polyethylene support (PES). Fermentation was performed in three cycles with refreshing the media at the beginning of each: cycle 1, 0-27 h; cycle 2, 27-54 h; and cycle 3, 54-78.5 h. Results showed that the glucose consumption and the pH reduction in the PES biofilm reactor was faster compared to the SSS biofilm reactor. Scanning electron microscopy showed that the SSS was capable to immobilize more cells during the growth of X. campestris. The maximum concentration of xanthan gum in the SSS biofilm reactor obtained after 27 h (3.47 ± 0.71 g/L), while the maximum concentration of xanthan in the PES biofilm reactor obtained after 78.5 h (3.21 ± 0.68 g/L). Thermal stability analysis of xanthan using differential scanning calorimetry showed the presence of two fractures attributed to dehydration and degradation of polymer. The thermogram represented both endothermal and exothermal behaviour of xanthan polymer. Furthermore, the functional groups and molecular structure of the xanthan produced in this study was evaluated using Fourier transform infrared spectrometry and also proton nuclear magnetic resonance. in addition, the surface tension of (0.2 %, w/v) xanthan gum solution was in a range of 52.16-56.5 mN/m. Rheological analysis of xanthan showed that the G' values were higher than the G″ in all frequencies demonstrating a relatively high elasticity of the produced xanthan gum., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Many animal and plant pathogenic bacteria employ a type three secretion system (T3SS) to deliver type three effector proteins (T3Es) into host cells. Efficient secretion of many T3Es in the plant pathogen Xanthomonas campestris pv. campestris (Xcc) relies on the global chaperone HpaB. However, how the domain of HpaB itself affects effector translocation/secretion is poorly understood. Here, we used genetic and biochemical approaches to identify a novel domain at the C-terminal end of HpaB (amino acid residues 137-160) that contributes to virulence and hypersensitive response (HR). Both in vitro secretion assay and in planta translocation assay showed that the secretion and translocation of T3E proteins depend on the C-terminal region of HpaB. Deletion of the C-terminal region of HpaB did not affect binding to T3Es, self-association or interaction with T3SS components. However, the deletion of C-terminal region sharply reduced the mounts of free T3Es liberated from the complex of HpaB with the T3Es, a reaction catalyzed in an ATP-dependent manner by the T3SS-associated ATPase HrcN. Our findings demonstrate the C-terminal domain of HpaB contributes to disassembly of chaperone-effector complex and reveal a potential molecular mechanism underpinning the involvement of HpaB in secretion of T3Es in Xcc., Competing Interests: The authors have declared that no competing interests exist.

Produced water (PW) is a by-product generated throughout oil exploration. Geological formation and geographical location of the reservoir influence its physical, chemical and biological characteristics. Xanthan gum (XG), an exopolysaccharide (EPS) produced by Xanthomonas campestris, has been widely used in enhanced oil recovery (EOR) technology because of its high viscosity, pseudoplastic behavior, stability in function of salinity, temperature and alkaline conditions. The production of XG may be affected by the composition of the PW, where the acetyl and pyruvyl radicals may be present in the mannoses. The aim of this study was to evaluate the composition of XG produced by X. campestris, particularly the amount of Xanthan, acetyl and pyruvyl groups, in culture mediums containing distilled (DW) or produced (PW) water in different concentrations, by means of dispersive Raman spectroscopy (1064 nm). The spectra of XG showed peaks referred to the main constituents of the Xanthan (glucose, mannose and glucuronic acid). Spectral features assigned to pyruvyl were seen in all samples mainly at ~1010 cm -1 , with higher intensity when using DW and 25% PW. PCA loadings showed that the peaks assigned to pyruvyl are consistent to presence of sodium pyruvate (~1040/~1050 and ~ 1432 cm -1 ) and were higher in the samples obtained in 25% PW. ANOVA GLM applied to Raman peaks of interest (~1010 and ~ 1090 cm -1 ) and to PCA scores (Score 1 to Score 3) showed that both were influenced by the type of water used in the culture medium, where the XG were strongly reduced in the groups PW compared to DW while the pyruvyl content increased proportionally with the concentration of PW. The results suggest that the composition of the water used in the bacteria's culture medium influenced the composition of XG, including the amount of Xanthan and particularly the pyruvyl content, and therefore needs to be considered when using this approach of injecting XG in oil fields as pyruvyl content affects viscosity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in crucifers. Our previous findings revealed that Xcc can degrade 4-hydroxybenzoic acid (4-HBA) via the β-ketoadipate pathway. This present study expands on this knowledge in several ways. First, we show that infective Xcc cells induce in situ biosynthesis of 4-HBA in host plants, and Xcc can efficiently degrade 4-HBA via the pobA/pobR locus, which encodes a 4-hydroxybenzoate hydroxylase and an AraC-family transcription factor respectively. Next, the transcription of pobA is specifically induced by 4-HBA and is positively regulated by PobR, which is constitutively expressed in Xcc. 4-HBA directly binds to PobR dimers, resulting in activation of pobA expression. Point mutation and subsequent isothermal titration calorimetry and size exclusion chromatography analysis identified nine key conserved residues required for 4-HBA binding and/or dimerization of PobR. Furthermore, overlapping promoters harboring fully overlapping -35 elements were identified between the divergently transcribed pobA and pobR. The 4-HBA/PobR dimer complex specifically binds to a 25-bp site, which encompasses the -35 elements shared by the overlapping promoters. Finally, GUS histochemical staining and subsequent quantitative assay showed that both pobA and pobR genes are transcribed during Xcc infection of Chinese radish, and the strain ΔpobR exhibited compromised virulence in Chinese radish. These findings suggest that the ability of Xcc to survive the 4-HBA stress might be important for its successful colonization of host plants., (© 2020 John Wiley & Sons Ltd.)

Lipopolysaccharides (LPSs) are microbe-associated molecular pattern molecules (MAMPs) from Gram-negative bacterial pathogens that potentially contain three different MAMPs (the O-polysaccharide chain, the oligosaccharide core and lipid A). LPSs was purified from Burkholderia cepacia, Pseudomonas syringae and Xanthomonas campestris and electrophoretically profiled. Outcomes of the interactions of the three different LPS chemotypes with Arabidopsis thaliana, as reflected in the induced defence metabolites, profiled at 12 h and 24 h post elicitation, were investigated. Plants were pressure-infiltrated with LPS solutions and methanol-based extractions at different time points were performed for untargeted metabolomics using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Multivariate data modelling and chemometric analysis were applied to generate interpretable biochemical information from the multidimensional data sets. The three LPSs triggered differential metabolome changes in the plants as apparent from chromatographically distinct MS chromatograms. Unsupervised and supervised multivariate data models exhibited time- and treatment-related variations, and revealed discriminating metabolite variables. Heat map models comparatively displayed the up-regulated pathways affecting the metabolomes and Venn diagrams indicated up-regulated and shared metabolites among the three LPS treatments. The altered metabolomes reflect the up-regulation of metabolites from not only the glucosinolate pathway, but also from the shikimate-phenylpropanoid-flavonoid -, terpenoid - and indolic/alkaloid pathways, as well as oxygenated fatty acids. Distinct phytochemical profiles, especially at the earlier time point, suggest differences in the perception of the three LPS chemotypes, associated with the molecular patterns within the tripartite lipoglycans., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Pathogens deploy effector proteins that interact with host proteins to manipulate the host physiology to the pathogen's own benefit. However, effectors can also be recognized by host immune proteins, leading to the activation of defence responses. Effectors are thus essential components in determining the outcome of plant-pathogen interactions. Despite major efforts to decipher effector functions, our current knowledge on effector biology is scattered and often limited. In this study, we conducted two systematic large-scale yeast two-hybrid screenings to detect interactions between Arabidopsis thaliana proteins and effectors from two vascular bacterial pathogens: Ralstonia pseudosolanacearum and Xanthomonas campestris. We then constructed an interactomic network focused on Arabidopsis and effector proteins from a wide variety of bacterial, oomycete, fungal, and invertebrate pathogens. This network contains our experimental data and protein-protein interactions from 2,035 peer-reviewed publications (48,200 Arabidopsis-Arabidopsis and 1,300 Arabidopsis-effector protein interactions). Our results show that effectors from different species interact with both common and specific Arabidopsis interactors, suggesting dual roles as modulators of generic and adaptive host processes. Network analyses revealed that effector interactors, particularly "effector hubs" and bacterial core effector interactors, occupy important positions for network organization, as shown by their larger number of protein interactions and centrality. These interactomic data were incorporated in EffectorK, a new graph-oriented knowledge database that allows users to navigate the network, search for homology, or find possible paths between host and/or effector proteins. EffectorK is available at www.effectork.org and allows users to submit their own interactomic data., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population-related behaviors in bacteria. However, the direct specific effect of QS molecules on host biology is largely understudied. In this work, we show that the QS molecule DSF ( cis -11-methyl-dodecenoic acid) produced by Xanthomonas campestris pv. campestris can suppress pathogen-associated molecular pattern-triggered immunity (PTI) in Arabidopsis thaliana , mediated by flagellin-induced activation of flagellin receptor FLS2. The DSF-mediated attenuation of innate immunity results from the alteration of FLS2 nanoclusters and endocytic internalization of plasma membrane FLS2. DSF altered the lipid profile of Arabidopsis , with a particular increase in the phytosterol species, which impairs the general endocytosis pathway mediated by clathrin and FLS2 nano-clustering on the plasma membrane. The DSF effect on receptor dynamics and host immune responses could be entirely reversed by sterol removal. Together, our results highlighted the importance of sterol homeostasis to plasma membrane organization and demonstrate a novel mechanism by which pathogenic bacteria use their communicating molecule to manipulate pathogen-associated molecular pattern-triggered host immunity., (© 2020 Tran et al.)

Homologous regulatory factors are widely present in bacteria, but whether homologous regulators synergistically or differentially regulate different biological functions remains mostly unknown. Here, we report that the homologous regulators RpoN1 and RpoN2 of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) play different regulatory roles with respect to virulence traits, flagellar biosynthesis, and basal metabolism. RpoN2 directly regulated Xcc fliC and fliQ to modulate flagellar synthesis in X. campestris, thus affecting the swimming motility of X. campestris. Mutation of rpoN2 resulted in reduced production of biofilms and extracellular polysaccharides in Xcc. These defects may together cause reduced virulence of the rpoN2 mutant against the host plant. Moreover, we demonstrated that RpoN1 could regulate branched-chain fatty acid production and modulate the synthesis of diffusible signal factor family quorum sensing signals. Although RpoN1 and RpoN2 are homologues, the regulatory roles and biological functions of these proteins were not interchangeable. Overall, our report provides new insights into the two different molecular roles that form the basis for the transcriptional specialization of RpoN homologues., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria translocates effector proteins via a type III secretion system (T3SS) into eukaryotic cells. The T3SS spans both bacterial membranes and consists of more than 20 proteins, 9 of which are conserved in plant and animal pathogens and constitute the core subunits of the secretion apparatus. T3S in X. campestris pv. vesicatoria also depends on nonconserved proteins with yet unknown function including HrpB7, which contains predicted N- and C-terminal coiled-coil regions. In the present study, we provide experimental evidence that HrpB7 forms stable oligomeric complexes. Interaction and localisation studies suggest that HrpB7 interacts with inner membrane and predicted cytoplasmic (C) ring components of the T3SS but is dispensable for the assembly of the C ring. Additional interaction partners of HrpB7 include the cytoplasmic adenosinetriphosphatase HrcN and the T3S chaperone HpaB. The interaction of HrpB7 with T3SS components as well as complex formation by HrpB7 depends on the presence of leucine heptad motifs, which are part of the predicted N- and C-terminal coiled-coil structures. Our data suggest that HrpB7 forms multimeric complexes that associate with the T3SS and might serve as a docking site for the general T3S chaperone HpaB., (© 2020 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Quorum quenching, which disrupts quorum sensing (QS) by either degradation of QS signals or interference of signal generation or perception, is a promising strategy for the prevention and control of QS-mediated bacterial infections. Diffusible signal factor (DSF) is widely conserved in many Gram-negative bacterial pathogens. In this study, we developed an efficient method for screening of highly active DSF degradation microorganisms. Among them, Pseudomonas sp. strain HS-18 showed a superior DSF degradation activity. Bioinformatics and genetic analyses showed that at least 4 genes, designated digA to digD , encoding fatty acyl coenzyme A ligase homologues, are responsible for DSF signal degradation. Interestingly, all 4 dig genes were induced by exogenous DSF, with digA being the most significantly induced. Expression of the dig genes in Xanthomonas campestris pv. campestris markedly reduced the accumulation of endogenous DSF, decreased production of virulence factors, and attenuated bacterial virulence on host plants. Similarly, application of strain HS-18 as a biocontrol agent could substantially reduce the disease severity caused by X. campestris pv. campestris These results unveil the molecular basis of a highly efficient DSF degradation bacterial isolate and present useful genes and biocontrol agents for control of the infectious diseases caused by DSF-dependent bacterial pathogens. IMPORTANCE Diffusible signal factor (DSF) represents a family of widely conserved quorum sensing signals involved in the regulation of virulence factor production in many Gram-negative bacterial pathogens. In this study, we developed a novel and efficient method for screening highly active DSF degradation microorganisms. With this method, we identified a bacterial isolate, Pseudomonas sp. strain HS-18, with a superb DSF degradation activity. We further found that strain HS-18 contains 4 genes responsible for DSF signal degradation, and significantly, these were induced by exogenous DSF molecules. These findings unveil the molecular basis of a highly efficient DSF degradation bacterial isolate and present useful methods, genes, and agents for control of the infectious diseases caused by DSF-dependent bacterial pathogens., (Copyright © 2020 American Society for Microbiology.)

Gamma-proteobacteria Xanthomonas spp. cause at least 350 different plant diseases among important agricultural crops, which result in serious yield losses. Xanthomonas spp. rely mainly on the type III secretion system (T3SS) to infect their hosts and induce a hypersensitive response in nonhosts. HrpG, the master regulator of the T3SS, plays the dominant role in bacterial virulence. In this study, we used chromatin immunoprecipitation followed by sequencing (ChIP-seq) and tandem affinity purification (TAP) to systematically characterize the HrpG regulon and HrpG interacting proteins in vivo. We obtained 186 candidate HrpG downstream genes from the ChIP-seq analysis, which represented the genomic-wide regulon spectrum. A consensus HrpG-binding motif was obtained and three T3SS genes, hpa2, hrcU, and hrpE, were confirmed to be directly transcriptionally activated by HrpG in the inducing medium. A total of 273 putative HrpG interacting proteins were identified from the TAP data and the DNA-binding histone-like HU protein of Xanthomonas campestris pv. campestris (HU xcc ) was proved to be involved in bacterial virulence by increasing the complexity and intelligence of the bacterial signalling pathways in the T3SS., (© 2020 Institute of Microbiology, Chinese Academy of Sciences. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

YajQ, a binding protein of the universal bacterial second messenger cyclic di-GMP (c-di-GMP), affects virulence in several bacterial pathogens, including Xanthomonas campestris. In this bacterium, YajQ interacts with the transcription factor LysR. Upon c-di-GMP binding, the whole c-di-GMP-YajQ-LysR complex is found to dissociate from DNA, resulting in virulence gene regulation. Here, we identify a YajQ-LysR-like system in the bacterial biocontrol agent Lysobacter enzymogenes OH11 that secretes an antifungal antibiotic, heat-stable antifungal factor (HSAF) against crop fungal pathogens. We show that the YajQ homologue, CdgL (c-di-GMP receptor interacting with LysR) affects expression of the HSAF biosynthesis operon by interacting with the transcription activator LysR. The CdgL-LysR interaction enhances the apparent affinity of LysR to the promoter region upstream of the HSAF biosynthesis operon, which increases operon expression. Unlike the homologues CdgL (YajQ)-LysR system in X. campestris, we show that c-di-GMP binding to CdgL seems to weaken CdgL-LysR interactions and promote the release of CdgL from the LysR-DNA complex, which leads to decreased expression. Together, this study takes the YajQ-LysR-like system from bacterial pathogens to a crop-protecting bacterium that is able to regulate antifungal HSAF biosynthesis via disassembly of the c-di-GMP receptor-transcription activator complex., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

A characteristic feature of phytopathogenic Xanthomonas bacteria is the production of yellow membrane-bound pigments called xanthomonadins. Previous studies showed that 3-hydroxybenzoic acid (3-HBA) was a xanthomonadin biosynthetic intermediate and also, that it had a signaling role. The question of whether the structural isomers 4-HBA and 2-HBA (salicylic acid) have any role in xanthomonadin biosynthesis remained unclear. In this study, we have selectively eliminated 3-HBA, 4-HBA, or the production of both by expression of the mhb , pobA , and pchAB gene clusters in the Xanthomonas campestris pv. campestris strain XC1. The resulting strains were different in pigmentation, virulence factor production, and virulence. These results suggest that both 3-HBA and 4-HBA are involved in xanthomonadin biosynthesis. When both 3-HBA and 4-HBA are present, X. campestris pv. campestris prefers 3-HBA for Xanthomonadin-A biosynthesis; the 3-HBA-derived Xanthomonadin-A was predominant over the 4-HBA-derived xanthomonadin in the wild-type strain XC1. If 3-HBA is not present, then 4-HBA is used for biosynthesis of a structurally uncharacterized Xanthomonadin-B. Salicylic acid had no effect on xanthomonadin biosynthesis. Interference with 3-HBA and 4-HBA biosynthesis also affected X. campestris pv. campestris virulence factor production and reduced virulence in cabbage and Chinese radish. These findings add to our understanding of xanthomonadin biosynthetic mechanisms and further help to elucidate the biological roles of xanthomonadins in X. campestris pv. campestris adaptation and virulence in host plants.

Background: Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein-DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid, and simplified Bind-n-seq protocol coupled with a user-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well-known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc)., Results: The improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes. Informatics analysis revealed that many of these genes regulate functions associated with virulence, motility, and biofilm formation and included genes previously found involved in virulence. Additionally, electromobility shift assays show that YipR binds to the promoter region of XC&#95;2633 in a CCCTCTC motif-dependent manner., Conclusion: We present a new and rapid Bind-n-seq protocol that should be useful to investigate DNA-binding proteins in bacteria. The analysis of YipR DNA binding using this protocol identifies a novel DNA sequence motif in the promoter regions of target genes that define the YipR regulon.

Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, produces a membrane-bound yellow pigment called xanthomonadin to protect against photobiological and peroxidative damage, and uses a quorum-sensing mechanism mediated by the diffusible signal factor (DSF) family signals to regulate virulence factors production. The Xcc gene XCC4003, annotated as Xcc fabG3, is located in the pig cluster, which may be responsible for xanthomonadin synthesis. We report that fabG3 expression restored the growth of the Escherichia coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrated that FabG3 catalyses the reduction of 3-oxoacyl-acyl carrier protein (ACP) intermediates in fatty acid synthetic reactions, although FabG3 had a lower activity than FabG1. Moreover, the fabG3 deletion did not affect growth or fatty acid composition. These results indicate that Xcc fabG3 encodes a 3-oxoacyl-ACP reductase, but is not essential for growth or fatty acid synthesis. However, the Xcc fabG3 knock-out mutant abolished xanthomonadin production, which could be only restored by wild-type fabG3, but not by other 3-oxoacyl-ACP reductase-encoding genes, indicating that Xcc FabG3 is specifically involved in xanthomonadin biosynthesis. Additionally, our study also shows that the Xcc fabG3-disrupted mutant affects Xcc virulence in host plants., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The two-component signalling system (TCS) comprising a histidine kinase (HK) and a response regulator (RR) is the predominant bacterial sense-and-response machinery. Because bacterial cells usually encode a number of TCSs to adapt to various ecological niches, the specificity of a TCS is in the centre of regulation. Specificity of TCS is defined by the capability and velocity of phosphoryl transfer between a cognate HK and a RR. Here, we provide genetic, enzymology and structural data demonstrating that the second messenger cyclic-di-GMP physically and specifically binds to RavS, a HK of the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. The [c-di-GMP]-RavS interaction substantially promotes specificity between RavS and RavR, a GGDEF-EAL domain-containing RR, by reinforcing the kinetic preference of RavS to phosphorylate RavR. [c-di-GMP]-RavS binding effectively decreases the phosphorylation level of RavS and negatively regulates bacterial swimming. Intriguingly, the EAL domain of RavR counteracts the above regulation by degrading c-di-GMP and then increasing the level of phosphorylated RavS. Therefore, RavR acts as a bifunctional phosphate sink that finely controls the level of phosphorylated RavS. These biochemical processes interactively modulate the phosphoryl flux between RavS-RavR and bacterial lifestyle transition. Our results revealed that c-di-GMP acts as an allosteric effector to dynamically modulate specificity between HK and RR., Competing Interests: The authors have declared that no competing interests exist.

The ability of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) to cause disease is dependent on its ability to adapt quickly to the host environment during infection. Like most bacterial pathogens, Xcc has evolved complex regulatory networks that ensure expression and regulation of their virulence genes. Here, we describe the identification and characterization of a Fis-like protein (named Flp), which plays an important role in virulence and type III secretion system (T3SS) gene expression in Xcc. Deletion of flp caused reduced virulence and hypersensitive response (HR) induction of Xcc and alterations in stress tolerance. Global transcriptome analyses revealed the Flp had a broad regulatory role and that most T3SS HR and pathogenicity (hrp) genes were down-regulated in the flp mutant. β-glucuronidase activity assays implied that Flp regulates the expression of hrp genes via controlling the expression of hrpX. More assays confirmed that Flp binds to the promoter of hrpX and affected the transcription of hrpX directly. Interestingly, the constitutive expression of hrpX in the flp mutant restored the HR phenotype but not full virulence. Taken together, the findings describe the unrecognized regulatory role of Flp protein that controls hrp gene expression and pathogenesis in Xcc., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Lignocellulosic-like materials are potentially low-cost fermentation substrates, but their pretreatment brings about by-products. This work investigated the effects of furfural on xanthan gum (XG) production, and product quality was evaluated by structure, viscosity and antioxidant capacities. Xanthomonas campestris maintained steady polysaccharide yield (above 13 g·L -1 ) with enhanced cell growth at low furfural concentrations (below 3.2 g·L -1 ). The products were verified as XG by FT-IR, XRD, NMR and monosaccharide analysis. Moreover, they were found to have reduced acetyl, rising pyruvate and up-to-down glucuronic acid groups as increasing furfural concentration. Furthermore, XG product with 1 g·L -1 furfural addition showed the best hydroxyl scavenging effects, though reducing powers presented no variation. It was demonstrated that furfural, the common hydrolysis by-product, was not necessarily an inhibitor for fermentation, and an appropriate amount of furfural was beneficial to XG production with steady yield and good quality., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In the present study, mixed and axenic submerged cultures of Cupriavidus necator and Xanthomonas campestris were performed for simultaneous and individual PHA and XG productions using palm oil (Elaeis guineensis) as substrate. Rotational Central Compound Design (RCCD) was successfully used in the optimization of individual productions of PHA (3.39 g L -1 , Mw = 692.6 kDa) and XG (1.77 g L -1 , Mw = 36.6 × 10 5  kDa). Novel simultaneous production of PHA (6.43 g L -1 , Mw = 629.2 kDa) and XG (1.98 g L -1 , Mw = 25.0 × 10 5  kDa), executed in bacterial co-cultivation, revealed to be a successful strategy to increment polymer synthesis, especially PHA. XG bioconversions followed a general trend of lower production in co-culture. Culture configurations also altered polymers properties and characteristics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Xanthan gum, whose structure determines its physicochemical properties, is an important microbial polysaccharide. Currently, marketed xanthan products are produced by wild-type strains followed by post-fermentation separation or chemical modification, which are complicated and labor-intensive. In the present study, we designed eight polysaccharides containing uniformly distributed repeating units and different rheological properties based on natural xanthan skeleton and according to the relationship between property and structure. The customized polysaccharides were produced in Xanthomonas campestris CGMCC 15155 using marker-less gene knockout and gene overexpression methods. The results showed that their different homogeneous primary structures determined their specific secondary structures and rheological properties, especially the terminal mannose, the pyruvyl group, and the acetyl group attached to the internal mannose of the side chain. Polysaccharides lacking a terminal mannose, such as xanthan XdM-0 and XdM-A, had reduced zero-shear viscosity and modulus values. The internal acetyl group of the side chain stabilized the helix structure (e.g., in XG-A0), while the pyruvate group had the opposite effect (e.g., in XG-AP and XG-0P). The eight xanthan variants provide a promising theoretical foundation to further study the structure-activity relationship of xanthan and will help to construct xanthan-containing block copolymers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

α-Arbutin is an effective skin-whitening cosmetic ingredient and can be synthesized through hydroquinone glycosylation. In this study, amylosucrase (Amy-1) from Xanthomonas campestris pv. campestris 8004 was newly identified as a sucrose-utilizing glycosylating hydroquinone enzyme. Its kinetic parameters showed a seven-time higher affinity to hydroquinone than maltose-utilizing α-glycosidase. The glycosylation of HQ can be quickly achieved with over 99% conversion when a high molar ratio of glycoside donor to acceptor (80:1) was used. A batch-feeding catalysis method was designed to eliminate HQ inhibition with high productivity (> 36.4 mM h -1 ). Besides, to eliminate the serious inhibition caused by the accumulated hydroquinone oxidation products, the whole-cell catalysis was further proposed. 306 mM of α-arbutin was finally achieved with 95% molar conversion rate within 15 h. Hence, the batch-feeding whole-cell biocatalysis by Amy-1 is a promising technology for α-arbutin production with enhanced yield and molar conversion rate.

Phenazine-1-carboxylic acid (PCA), a secondary metabolite produced by Pseudomonas spp., exhibits a high inhibitory effect in Xanthomonas oryzae pv. oryzae (Xoo), but less inhibitory effect in Xanthomonas oryzae pv. oryzicola (Xoc), and almost no inhibitory effect in Xanthomonas campestris pv. campestris (Xcc). In our previous study, reactive oxygen species (ROS) scavenging system was reported to be involved in PCA tolerance in Xanthomonas spp. However, the PCA tolerance mechanism of Xanthomonas spp. is unclear. In the current study, we constructed a Tn5-based transposon mutant library in Xcc and four highly PCA-sensitive insertion mutants were obtained. TAIL-PCR further confirmed that the Tn5 transposon was inserted in the cytochrome c maturation (CCM) system (XC&#95;1893, XC&#95;1897) of these mutants. Disruption of the CCM system significantly decreased the growth, motility and tolerance of Xcc to PCA and other phenazines, such as phenazine and 1-OH-phenazine. The CCM system is responsible for the covalent attachment of the apocytochrome and heme. Disruption of the transmembrane thioredox protein (Dsb) pathway (XC&#95;0531), an essential process for the formation of mature apocytochrome, also exhibited a decreased tolerance to PCA, suggesting that the defect of cytochrome c caused decreased tolerance of Xcc to PCA. Meanwhile, disruption of the CCM system or Dsb pathway interfered with the functions of cytochrome c proteins, causing an increased sensitivity to H 2 O 2 . Collectively, we concluded that the CCM system and Dsb pathway, regulate the tolerance of Xcc to phenazines by influencing the functions of cytochrome c. Therefore, these results provide important references for revealing the action mechanism of PCA in Xanthomonas spp., (Copyright © 2019 Elsevier Inc. All rights reserved.)