Functional analysis of the role of Fur in the virulence of Pseudomonas syringae pv. tabaci 11528: Fur controls expression of genes involved in quorum-sensing

doi:10.1016/j.bbrc.2007.11.021

Biochemical and Biophysical Research Communications

Volume 366, Issue 2, 8 February 2008, Pages 281-287

https://doi.org/10.1016/j.bbrc.2007.11.021 Get rights and content

Abstract

In most bacteria, Fur (ferric uptake regulator) is a crucial global regulator known to operate not only in the regulation of iron homeostasis but also in a variety of other cellular processes. In an effort to characterize the role of Fur in the virulence of plant pathogens, a fur homolog was isolated from Pseudomonas syringae pv. tabaci 11528. Phenotype assays showed that a fur deletion mutant (BL33) constitutively produced siderophore(s) and exhibited decreases in swarming motility as well as the synthesis of tabtoxin and N-acyl homoserine lactones. Consistent with the results of TLC, quantitative real-time RT-PCR of the QS associated genes psyR and psyI demonstrated that Fur up-regulates these genes at the transcriptional level. Finally, the effects of a fur mutation on plant virulence indicated that Fur-regulated traits are relevant to plant–pathogen interactions.

Section snippets

Materials and methods

Bacterial strains, plasmids, and growth conditions. All bacterial strains and plasmids used in this study are listed in Table 1. P. syringae pv. tabaci strains were routinely grown in King’s B (KB) [23] medium at 30 °C. Siderophore production was studied by the addition of either 50 μM FeCl₃ or 100 μM 2,2′-dipyridyl to chrome azure S (CAS) plates to achieve high- or low-iron conditions, respectively [1]. Antibiotics were added to the media at the following concentrations (μg/ml): ampicillin, 50;

Identification and analysis of a fur homolog in P. syringae pv. tabaci 11528

In our attempt to identify a fur homolog in P. syringae pv. tabaci 11528, specific primers (Table 2) were used to PCR-amplify a 1513 bp genomic DNA fragment. Sequence analysis of the DNA fragment, cloned into pRK415 (pBL177), revealed a 408-bp ORF with a high degree of homology to the fur genes of other P. syringae pathovars. This ORF encoded a 135 amino acid, histidine-rich (7%) protein with a calculated molecular mass of 15,241 Da and a pI of 5.30. The predicted amino acid sequence indicated

Acknowledgments

This work was supported by the Brain Korea 21 program of the Korean Ministry of Education (Brain Korea 21 program) and for two years by a Pusan National University Research Grant.

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Bacterial leaf streak disease caused by Xanthomonas oryzae pv. Oryzicola (Xoc) is a serious threat to rice production worldwide. Like most bacterial phytopathogens, Xoc also contains multiple regulators involved in the complex regulatory networks to ensure its virulence. In this study, we found two genes, FE36_09860 and FE36_06255, through quantitative fluorescence PCR essential for Xoc virulence and might be regulated by the diffusible signal factor (DSF) and the global regulator (Clp). We evaluated the characteristics of the TetR family (FE36_09860) and Fur family (FE36_06255) transcriptional regulator genes in in-vitro and in-vivo experiments. Results demonstrated that the deletion of two transcriptional regulators genes, FE36_09860 and FE36_06255, reduced the virulence of Xoc. Further analysis revealed that the deletion of the FE36_06255 gene results in decreased EPS production, elevated biofilm formation and reactive oxygen species production, and maintained the growth rate of Xoc YM15. In contrast, the deletion of FE36_09860 showed an opposite trend. It is concluded that, as transcriptional regulators, the FE36_09860 and FE36_06255 genes played an important role in the biological processes of Xoc YM15 and were essential for its virulence.
Virulence-related regulatory network of Pseudomonas syringae
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Citation Excerpt :
Fur acts as an iron-responsive repressor by binding to its target gene elements, known as Fur box [89]. In iron-limited or iron-rich conditions, Fur slightly and strongly represses siderophore production [88]. In addition, GacAS and SalA play roles in the modulation of siderophore-related genes.
Transcription factors (TFs) play important roles in regulating multiple biological processes by binding to promoter regions and regulating the global gene transcription levels. Pseudomonas syringae is a Gram-negative phytopathogenic bacterium harbouring 301 putative TFs in its genome, approximately 50 of which are responsible for virulence-related gene and pathway regulation. Over the past decades, RNA sequencing, chromatin immunoprecipitation sequencing, high-throughput systematic evolution of ligands by exponential enrichment, and other technologies have been applied to identify the functions of master regulators and their interactions in virulence-related pathways. This review summarises the recent advances in the regulatory networks of TFs involved in the type III secretion system (T3SS) and non-T3SS virulence-associated pathways, including motility, biofilm formation, quorum sensing, nucleotide-based secondary messengers, phytotoxins, siderophore production, and oxidative stress. Moreover, this review discusses the future perspectives in terms of TF-mediated pathogenesis mechanisms and provides novel insights that will help combat P. syringae infections based on the regulatory networks of TFs.
Contributions of ferric uptake regulator Fur to the sensitivity and oxidative response of Acinetobacter baumannii to antibiotics
2018, Microbial Pathogenesis
Citation Excerpt :
It was proposed that generated H2O2 undergo Fenton reaction in the presence of Fe2+ leading to hydroxyl radical production, DNA damage and bacterial death [10,11,24]. Furthermore, fur has been demonstrated to regulate oxidative stress and virulence [29,30]. We hypothesized that the reduced SOD and CAT activity observed in Δfur mutant should lead to ROS accumulation.
Ferric uptake regulator (Fur) is important in the regulation of bacterial iron metabolism and uptake of Fe from the environment. We evaluated the contribution of fur to the sensitivity and oxidative response of A. baumannii to antibiotics. Deletion of fur increased the sensitivity of A. baumannii AB5075 to colistin, gentamicin, rifampicin and tigecycline. Furthermore, activities of superoxide dismutase and catalase in Δfur mutant decreased significantly compared to the parental strain. Conversely, •O₂− and H₂O₂ accumulate in colistin, gentamicin, rifampicin or tigecycline-treated Δfur mutant compared to the parental strain. Ferrous ion (Fe²⁺) content of Δfur mutant increased compared to the parental strain. Fe chelator 2,2′-bipyridyl lowered the sensitivity of A. baumannii to the antibiotics. The antibiotics, except tigecycline, raised the NAD+/NADH and ADP/ATP ratio of Δfur mutant compared to the WT. Glutathione content of Δfur mutant was significantly depleted compared to parental strain following exposure to the antibiotics. We conclude that decreased capability of Δfur mutant to detoxify reactive oxygen species raised its susceptibility to antibiotics through Fenton chemistry.
Lag phase-associated iron accumulation is likely a microbial counter-strategy to host iron sequestration: Role of the ferric uptake regulator (fur)
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Citation Excerpt :
As Fur copies increase, ferritin concentration would increase as well, consequently transferring accumulated ‘free’ iron to the ‘stored’ state within ferritin whilst ameliorating the oxidative impact of iron. It is also notable that Fur is implicated in virulence because Δfur strains display reduced efficiency in colonization and reduced infectivity in several infection models (Bury-Moné et al., 2004; Gancz et al., 2006; Cha et al., 2008; Horsburgh et al., 2001; Rea et al., 2004; Jacobsen et al., 2005; Harrison et al., 2013). Constitutive de-regulation of antioxidant enzymes, iron uptake and storage proteins, as well as virulence proteins, most probably results in multiple deleterious phenotypes (Touati et al., 1995; Benov and Sequeira, 2003; Hoerter et al., 2005; Jacobsen et al., 2005).
Iron is an essential metal for almost all forms of life, but potentiates oxidative stress via Fenton catalysis. During microbial lag phase there is a rapid influx of iron with concomitant oxidative hypersensitivity. How and why iron accumulation occurs remains to be elucidated. Iron homeostasis in prokaryotes is mediated by the ferric uptake regulator (Fur), an iron-activated global regulator that controls intracellular iron levels by feedback inhibition with the metal. Herein it is postulated, based on the expression profiles of antioxidant enzymes within the Fur regulon as observed in wild type and Δfur mutants, that iron accumulation is mediated by a transitively low concentration of the Fur protein during lag phase. Vertebrate hosts sequester iron upon ‘sensing’ an infection in order to retard microbial proliferation through a process known as ‘nutritional immunity’. It is herein argued that the purpose of iron accumulation is not principally a preparative step for the replicative phase, as suggested elsewhere, but an evolved behavior that counteracts host iron sequestration. This interpretation is supported by multiple clinical and animal studies that demonstrate that iron surplus in hosts advances progression and susceptibility to infection, and vice versa. Contextualizing iron accumulation as a counter-immune behavior adds impetus to the development of antibiotics targeting pathogenic modes of iron acquisition.
GacA directly regulates expression of several virulence genes in Pseudomonas syringae pv. tabaci 11528
2012, Biochemical and Biophysical Research Communications
Citation Excerpt :
A 1147-bp PCR fragment was cloned into a pGEM-T Easy vector (Promega, USA) and a 202-bp fragment internal to gacA was deleted by digestion and relegation, resulting in pBL190. The 945-kb XbaI-SalI fragment of pBL190 was then subcloned into the suicide vector pLO1 [10], to create pBL111. The regions flanking the prhI locus of P. syringae pv.
A two-component system comprising GacS and GacA affects a large number of traits in many Gram-negative bacteria. However, the signals to which GacS responds, the regulation mechanism for GacA expression, and the genes GacA controls are not yet clear. In this study, several phenotypic tests and tobacco-leaf pathogenicity assays were conducted using a gacA deletion mutant strain (BL473) of Pseudomonas syringae pv. tabaci 11528. To determine the regulation mechanism for gacA gene expression and to identify GacA-regulated genes, we conducted quantitative RT-PCR and electrophoretic mobility shift assay (EMSA) experiments. The results indicated that virulence traits related to the pathogenesis of P. syringae pv. tabaci 11528 are regulated coordinately by GacA and iron availability. They also revealed that several systems coordinately regulate gacA gene expression in response to iron concentration and bacterial cell density and that GacA and iron together control the expression of several virulence genes. EMSA results provided genetic and molecular evidence for direct control of virulence genes by GacA.
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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Identification and analysis of a fur homolog in P. syringae pv. tabaci 11528

Acknowledgments

References (25)

Universal chemical assay for the detection and determination of siderophores

Anal. Biochem.

The role of iron in plant host–pathogen interactions

Trends Microbiol.

Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria

Curr. Opin. Microbiol.

Characterization of a ferric uptake regulator (fur) gene from Xanthomonas campestris pv. phaseoli with unusual primary structure, genome organization, and expression patterns

Gene

Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria

Gene

Quorum sensing regulates exopolysaccharide production, motility, and virulence in Pseudomonas syringae

Mol. Plant-Microbe Interact.

Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa

J. Bacteriol.

Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator

Mol. Microbiol.

Role of the Fur regulon in iron transport in Bacillus subtilis

J. Bacteriol.

Molecular Cloning: A Laboratory Manual

Bradyrhizobium japonicum senses iron through the status of haem to regulate iron homeostasis and metabolism

Mol. Microbiol.

Quorum sensing in bacteria

Annu. Rev. Microbiol.

Cited by (41)

Identification and characterization of two transcriptional regulators in Xanthomonas oryzae pv. oryzicola YM15

Virulence-related regulatory network of Pseudomonas syringae

Contributions of ferric uptake regulator Fur to the sensitivity and oxidative response of Acinetobacter baumannii to antibiotics

Lag phase-associated iron accumulation is likely a microbial counter-strategy to host iron sequestration: Role of the ferric uptake regulator (fur)

GacA directly regulates expression of several virulence genes in Pseudomonas syringae pv. tabaci 11528

The Role of Iron in Phytopathogenic Microbe–Plant Interactions: Insights into Virulence and Host Immune Response