Enhanced production of 2-hydroxyphenazine in <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> GP72

Pseudomonas chlororaphis GP72 is a root-colonizing biocontrol strain isolated from the green pepper rhizosphere that synthesizes two phenazine derivatives: phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). The 2-OH-PHZ derivative shows somewhat stronger broad-spectrum antifungal activity than PCA, but its conversion mechanism has not yet been clearly revealed. The aim of this study was to clone and analyze the phenazine biosynthesis gene cluster in this newly found strain and to improve the production of 2-OH-PHZ by gene disruption and precursor addition. The conserved phenazine biosynthesis core operon in GP72 was cloned by PCR, and the unknown sequences located upstream and downstream of the core operon were detected by random PCR gene walking. This led to a complete isolation of the phenazine biosynthesis gene cluster phzIRABCDEFG and phzO in GP72. Gene rpeA and phzO were insertionally mutated to construct GP72AN and GP72ON, respectively, and GP72ANON collectively. The inactivation of rpeA resulted in a fivefold increase in the production of PCA, as well as 2-OH-PHZ. The addition of exogenous precursor PCA to the broth culture, to determine the conversion efficiency of PCA to 2-OH-PHZ under current culture conditions, revealed that PCA had a positive feedback effect on its own accumulation, leading to enhanced synthesis of both PCA and 2-OH-PHZ. The production of 2-OH-PHZ by GP72AN increased to about 170 μg ml⁻¹, compared with just 5 μg ml⁻¹ for the wild type. The hypothesis of biosynthetic pathway for 2-OH-PHZ from PCA was confirmed by identification of 2-hydroxyphenazine-1-carboxylic acid as an intermediate in the culture medium of the high-phenazine producing GP72AN mutant.     

GacS-dependent regulation of enzymic and antifungal activities and synthesis of <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones in rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

Pseudomonas chlororaphis strain 449 isolated from the rhizosphere of maize suppresses numerous plant pathogens in vitro. The strain produces phenazine antibiotics and synthesizes at least three types of quorum sensing signaling molecules, N-acylhomoserine lactones. Here we have shown that the rhizospheric P. chlororaphis strains 449, well known strain 30–84 as well as two other P. chlororaphis strains exhibit polygalacturonase activity. Using mini-Tn5 transposon mutagenesis, four independent mutants of strain P. chlororaphis 449 with insertion of mini-Tn5 Km2 in gene gacS of two-component GacA-GacS system of global regulation were selected. All these mutant strains were deficient in production of extracellular proteinase(s), phenazines, N-acylhomoserine lactones synthesis, and did not inhibit the growth of G⁺ bacteria in comparison with the wild type strain. The P. chlororaphis 449-06 gacS ⁻ mutant studied in greater detail was deficient in polygalacturonase, pectin methylesterase activities, swarming motility and antifungal activity. It is the first time the involvement of GacA-GacS system in the regulation of enzymes of pectin metabolism, polygalacturonase and pectin methylesterase, was demonstrated in fluorescent pseudomonads.     

Role of alternative sigma factor 54 (RpoN) from Vibrio anguillarum M3 in protease secretion, exopolysaccharide production, biofilm formation, and virulence

The sigma factor σ⁵⁴ (RpoN) is an important regulator of bacterial response to environmental stresses. Here, we demonstrate the roles of RpoN in Vibrio anguillarum M3 by comparative investigation of physiological phenotypes and virulence of the wild-type, an rpoN mutant, and an rpoN complemented strain. Disruption of rpoN was found to decrease biofilm formation, production of exopolysaccharides, and production of the metalloproteases EmpA and PrtV. Injection experiments in fish showed that the M3 ΔrpoN mutant was attenuated in virulence when administrated either by intramuscular injection or by immersion challenge. Slower proliferation of the mutant in fish was also observed. Complementation of the mutant strain with rpoN restored some of the phenotypes to wild-type levels. RpoN was involved in regulation of some virulence-associated genes, as shown by real-time quantitative reverse PCR analysis. These results revealed a pleiotropic regulatory role of RpoN in biofilm formation, production of proteases and exopolysaccharides, and virulence in V. anguillarum M3.     

Differential Regulation of rsmA Gene on Biosynthesis of Pyoluteorin and Phenazine-1-carboxylic Acid in Pseudomonas sp. M18

Summary Plant growth promoting rhizobacteria (PGPR) strain Pseudomonas sp. M18 can produce two different types of antibiotics, pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA). The global regulator RsmA is a translational repressor of secondary metabolism in many prokaryotes. A chromosomally rsmA inactivated mutant strain M18R was constructed to study the regulatory mechanism of Plt and PCA biosynthesis and enhancement of Plt or PCA production in Pseudomonas sp. M18. The accumulation of Plt increased six-fold over that of the wild-type strain whereas PCA production was not significantly affected in cultures of M18R. Plt production was inhibited completely but PCA biosynthesis was not altered after complementation with rsmA gene in trans in the strain of M18R. The differential activity of rsmA gene on these two operons was further confirmed by the analysis of β-galactosidase activities from translational phzA′-′lacZ and pltA′-′lacZ fusion, in which phzA is the first enzyme gene of the phenazine biosynthesis pathway and pltA is the first gene of the pyoluteorin biosynthesis pathway. The results indicate that RsmA can control Plt production negatively but not PCA production in M18, and show that the global regulator RsmA does not repress the biosynthesis of all secondary metabolites.     

Nucleotide sequence of the rpoN (hno) gene region of Alcaligenes eutrophus: evidence for a conserved gene cluster

The nucleotide sequence of the rpoN gene, formerly designated hno, and flanking DNA regions of the aerobic hydrogen bacterium Alcaligenes eutrophus has been determined; rpoN codes for the RNA polymerase sigma factor ⁵⁴ involved in nitrogen regulation and diverse physiological functions of gram-negative bacteria. In A. eutrophus hydrogen metabolism is under control of rpoN. The Tn5-Mob insertion in a previously isolated pleiotropic mutant was mapped within the rpoN gene. The derived amino acid sequence of the A. eutrophus RpoN protein shows extensive homology to the RpoN proteins of other organisms. Sequencing revealed four other open reading frames: one upstream (ORF280) and three downstream (ORF130, ORF99 and ORF > 54) of the rpoN gene. A similar arrangement of homologous ORFs is found in the rpoN regions of other bacteria and is indicative of a conserved gene cluster.     

Proteomic based investigation of rhamnolipid production by Pseudomonas chlororaphis strain NRRL B-30761

We recently reported that a strain of the non-pathogenic bacterial species Pseudomonas chlororaphis was capable of producing the biosurfactant molecule, rhamnolipids. Previous to this report the organisms known to produce rhamnolipids were almost exclusively pathogens. The newly described P. chlororaphis strain produced rhamnolipids at room temperature in static minimal media, as opposed to previous reports of rhamnolipid production which occurred at elevated temperatures with mechanical agitation. The non-pathogenic nature and energy conserving production conditions make the P. chlororaphis strain an attractive candidate for commercial rhamnolipid production. However, little characterization of molecular/biochemical processes in P. chlororaphis have been reported. In order to achieve a greater understanding of the process by which P. chlororaphis produces rhamnolipids, a survey of proteins differentially expressed during rhamnolipid production was performed. Separation and measurement of the bacteria’s proteome was achieved using Beckman Coulter’s Proteome Lab PF2D packed column-based protein fractionation system. Statistical analysis of the data identified differentially expressed proteins and known orthologues of those proteins were identified using an AB 4700 Proteomics Analyzer mass spectrometer system. A list of proteins differentially expressed by P. chlororaphis strain NRRL B-30761 during rhamnolipid production was generated, and confirmed through a repetition of the entire separation process.Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Optimization of nutrient components for enhanced phenazine-1-carboxylic acid production by <Emphasis Type="Italic">gacA</Emphasis>-inactivated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18G using response surface method

The nutritional requirements for phenazine-1-carboxylic acid (PCA) production using Pseudomonas sp. M18G, a gacA chromosomal-inactivated mutant of the strain M18, with a high PCA yield, were optimized statistically in shake flask experiments. Based on a single-factor experiment design, we implemented the two-level Plackett–Burman (PB) design with 11 variables to screen medium components that significantly influence PCA production. Soybean meal, glucose, soy peptone, and ethanol were identified as the most important significant factors (P < 0.05). Response surface methodology based on the Center Composite Design (CCD) was applied to determine these factors’ optimal levels and their mutual interactions between components for PCA production. The predicted results showed that 1.89 g l⁻¹ of PCA production was obtained after a 60-h fermentation period, with optimal concentrations of soybean meal powder (33.4 g l⁻¹), glucose (12.7 g l⁻¹), soy peptone (10.9 g l⁻¹), and ethanol (13.8 ml l⁻¹) in the flask fermentations. The validity of the model developed was verified, and the optimum medium led to a maximum PCA concentration of 2.0 g l⁻¹, a nearly threefold increase compared to that in the basal medium. Furthermore, the experiment was scaled up in the 10 l fermentor and 2 g l⁻¹ PCA productions were achieved in 48 h based on optimization mediums which further verified the practicability of this optimum strategy.     

Effects of kinetin and nitrogen on growth rates,pigment and protein contents in wild and phycoerythrin-deficient strains of <Emphasis Type="Italic">Hypnea musciformis</Emphasis> (Rhodophyta)

Hypnea musciformis (Wulfen in Jacqu.) J.V. Lamour. is the main source for carrageenan production in Brazil and strains with selected characteristics could improve the production of raw material. The effects of kinetin on growth rates, morphology, protein content, and concentrations of pigments (chlorophyll a, phycoerythrin, phycocyanin, and allophycocyanin) were assessed in the wild strain (brown phenotype) and in the phycoerythrin-deficient strain (green phenotype) of H. musciformis. Concentrations of kinetin ranging from 0 to 50 μM were tested in ASP 12-NTA synthetic medium with 10 μM nitrate (N-limited) and 100 μM nitrate (N-saturated). In N-limited condition, kinetin stimulated growth rates of the phycoerythrin-deficient strain and formation of lateral branches in both colour strains. Kinetin stimulated protein biosynthesis in both strains. However, differences between both nitrogen conditions were significant only in the phycoerythrin-deficient strain. In the wild strain, effects of kinetin on concentrations of phycobiliproteins were not significant in both nitrogen conditions, except for chlorophyll content. However, the phycoerythrin-deficient strain showed an opposite response, and kinetin stimulated the phycobiliprotein biosynthesis, with the highest concentrations of phycoerythrin in N-saturated medium, while the highest concentrations of allophycocyanin and phycocyanin were observed in N-limited medium. These results indicate that the effects of kinetin on growth, morphology, protein and phycobiliprotein contents are influenced by nitrogen availability, and the main nitrogen storage pools in phycoerythrin-deficient strain of H. musciformis submitted to N-limited conditions were phycocyanin and allophycocianin, the biosynthesis of which was enhanced by kinetin.     

Structure and expression of the alternative sigma factor, RpoN, in Rhodobacter capsulatus; physiological relevance of an autoactivated nifU2-rpoN superoperon

The alternative sigma factor, RpoN (σ⁵⁴) is responsible for recruiting core RNA polymerase to the promoters of genes required for diverse physiological functions In a variety of eubacterial species. The RpoN protein In Rhodobacter capsulatus is a putative sigma factor specific for nitrogen fixation (nif) genes. Insertional mutagenesis was used to define regions important for the function of the R. capsulatus RpoN protein. Insertions of four amino acids in the predicted helix-turn-helix or in the highly conserved C-terminal eight amino acid residues (previously termed the RpoN box), and an in-frame deletion of the glutamine-rich M-terminus completely inactivated the R. capsulatus RpoN protein. Two separate insertions in the second hydrophobic heptad repeat, a putative leucine zipper, resulted in a partially functional RpoN protein. Eight other linkers in the rpoN open reading frame (ORF) resulted in a completeiy or partially functional RpoN protein. The rpoN gene in R capsulatus is downstream from the nifHDKU2 genes, in a nifU2-rpoN operon. Results of genetic experiments on the nifU2-rpoN locus show that the rpoN gene is organized in a nifU2-rpoN superoperon. A primary promoter directly upstream of the rpoN ORF is responsible for the initial expression of rpoN. Deletion analysis and insertional mutagenesis were used to define the primary promoter to 50 bp, between 37 and 87 nucleotides upstream of the predicted rpoN translational start site. This primary promoter is expressed constitutively with respect to nitrogen, and it is necessary and sufficient for growth under nitrogen-limiting conditions typically used in the laboratory. A secondary promoter upstream of nifU2 is autoactivated by RpoN and NifA to increase the expression of rpoN, which ultimately results in higher expression of RpoN dependent genes. Moreover. rpoN expression from this secondary promoter is physiologically beneficial under certain stressful conditions, such as nitrogen-limiting environments that contain high salt (>50mM NaCl) or low iron (<400nM FeS0₄).     

OxyR,an important oxidative stress regulator to phenazines production and hydrogen peroxide resistance in Pseudomonas chlororaphis GP72

Pseudomonas chlororaphis GP72 is an important plant growth-promoting rhizobacteria (PGPR) with a wide-spectrum antibiotic activity toward several soil-borne pathogens. The adaption of this strain to different environmental oxidative stress and redox phenazine pigment by the predicted regulator OxyR were investigated. The deletion of oxyR led to a significant reduction of the viability, production of three phenazine derivatives and resistance to hydrogen peroxide and paraquat on the KB agar plates. However, the mutant ΔoxyR grew better with shorter delay. In addition, the mutant ΔoxyR showed an increased resistance to hydrogen peroxide, which occurred at the concentration varying from 1.0 mM to 5.0 mM in the KB broth, as compared with the wild type. In addition, the biofilm formation ability was obviously enhanced and influenced by the different oxidants in the mutant. Quantitative RT-PCR experiments indicated that the expression of katG, ahpC, ahpD and phzE were increased in the oxyR mutant background in response to hydrogen peroxide. katG was mainly responsible for the enhanced resistance to hydrogen peroxide. The loss of oxyR is suggested to benefit the hydrogen peroxide inducible gene expression. Thus, OxyR is an important global regulator that regulates multiple pathways to enhance the survival of P. chlororaphis GP72 exposed to different oxidative stresses.     

Utilization of <Emphasis Type="Italic">fadA</Emphasis> knockout mutant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> for overproduction of medium chain-length-polyhydroxyalkanoate

The fadBA operon in the fatty acid β-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA). Succinate at 150 mg l⁻¹ stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l⁻¹ with a cell dry weight of 10.3 g l⁻¹.     

Production of Indole-3-Acetic Acid in the Plant-Beneficial Strain Pseudomonas chlororaphis O6 Is Negatively Regulated by the Global Sensor Kinase GacS

Certain plant growth–promoting bacteria, such as Pseudomonas fluorescens 89B61 and Bacillus pumilus SE34, secreted high levels of indole-3-acetic acid (IAA) in tryptophan-amended medium in stationary phase as determined by chromogenic analysis and high-performance liquid chromatography. Two other growth-promoting strains, P. chlororaphis O6 and Serratia marcescens 90-166, did not produce these high levels of IAA. However, when the gacS mutant of P. chlororaphis O6 was grown in tryptophan-supplemented medium, IAA was detected in culture filtrates. IAA production by the gacS mutant in P. chlororaphis O6 was repressed in the tryptophan medium by complementation with the wild-type gacS gene. Thus, the global regulatory Gac system in P. chlororaphis O6 acts as a negative regulator of IAA production from trypophan.     

Characterization of a Phenazine-Producing Strain Pseudomonas chlororaphis GP72 with Broad-Spectrum Antifungal Activity from Green Pepper Rhizosphere

A new Pseudomonas strain, designated GP72, was isolated from green pepper rhizosphere and identified as a member of species Pseudomonas chlororaphis based on morphology; conventional biochemical and physiologic tests; Biolog GN system (Biolog Inc., Hayward, CA); and 16S rDNA sequence analysis. The secondary metabolites produced by this strain have shown broad-spectrum antifungal activity against various phytopathogens of agricultural importance in vitro. Two main antifungal substances produced by this strain proved to be phenazine-1-carboxylic acid and 2-hydroxyphenazine with further purification and structure elucidation based on ultraviolet-absorbent spectrum scanning, atmospheric pressure chemical ionization–mass spectrometry (APCI-MS) spectrum, and ¹H,¹³C nuclear magnetic resonance spectrums. Strain GP72 could produce quorum-sensing signaling molecules of N-butanoyl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone, which were found to accumulate with different quantities in King’s medium B and pigment producing medium, respectively.     

Characterization of new isolated <Emphasis Type="Italic">Ralstonia</Emphasis><Emphasis Type="Italic">eutropha</Emphasis> strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) production

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and γ–hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of γ–hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments. The nucleotide sequence 1,378 bp reported in this study will appear in the GenBank nucleotide sequence database under accession number EF988626.     

The alternative sigma factors,rpoN1 and rpoN2 are required for mycophagous activity of Burkholderia gladioli strain NGJ1

Bacteria utilize RpoN, an alternative sigma factor (σ54) to grow in diverse habitats, including nitrogen-limiting conditions. Here, we report that a rice-associated mycophagous bacterium Burkholderia gladioli strain NGJ1 encodes two paralogues of rpoN viz. rpoN1 and rpoN2. Both of them are upregulated during 24 h of mycophagous interaction with Rhizoctonia solani, a polyphagous fungal pathogen. Disruption of either one of rpoNs renders the mutant NGJ1 bacterium defective in mycophagy, whereas ectopic expression of respective rpoN genes restores mycophagy in the complementing strains. NGJ1 requires rpoN1 and rpoN2 for efficient biocontrol to prevent R. solani to establish disease in rice and tomato. Further, we have identified 17 genes having RpoN regulatory motif in NGJ1, majority of them encode potential type III secretion system (T3SS) effectors, nitrogen assimilation, and cellular transport-related functions. Several of these RpoN regulated genes as well as certain previously reported T3SS apparatus (hrcC and hrcN) and effector (Bg_9562 and endo-β-1,3-glucanase) encoding genes are upregulated in NGJ1 but not in ΔrpoN1 or ΔrpoN2 mutant bacterium, during mycophagous interaction with R. solani. This highlights that RpoN1 and RpoN2 modulate T3SS, nitrogen assimilation as well as cellular transport systems in NGJ1 and thereby promote bacterial mycophagy.     

Th1-type immune response to infection by pYV-cured <Emphasis Type="Italic">phoP-phoQ</Emphasis> null mutant of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> is defective in mouse model

The PhoP-PhoQ two-component system of Yersinia pseudotuberculosis, a Gram-negative enteric pathogen which causes a variety of gastrointestinal and extraintestinal infections in humans, has been shown to be necessary for virulence. A phoP-phoQ null mutant of a strain of Y. pseudotuberculosis cured of its native plasmid pYV was obtained and studied for generation of immune response in mouse model following intravenous inoculation. The phoP-phoQ null mutant elicited much weaker IgG antibody response to whole cell sonicated (WCS) antigen, in particular that of IgG2a isotype. Interferon-γ levels were also significantly reduced in cultured splenocytes of mice immunized with phoP-phoQ null mutant. The null mutant was found to be about 72-fold less virulent than the parent isogenic strain of Y. pseudotuberculosis. Average counts in spleen of mice inoculated with the null mutant were observed to reduce by at least four logs when compared with the counts in the spleen of mice inoculated with parent isogenic strain. We can thus suggest that the Th1-type immune response of the phoP-phoQ null mutant of Y. pseudotuberculosis is diminished in mice.     

The fire blight pathogen Erwinia amylovora requires the rpoN gene for pathogenicity in apple

RpoN is a σ⁵⁴ factor regulating essential virulence gene expression in several plant pathogenic bacteria, including Pseudomonas syringae and Pectobacterium carotovorum. In this study, we found that mutation of rpoN in the fire blight pathogen Erwinia amylovora caused a nonpathogenic phenotype. The E. amylovora rpoN Tn5 transposon mutant rpoN1250::Tn5 did not cause fire blight disease symptoms on shoots of mature apple trees. In detached immature apple fruits, the rpoN1250::Tn5 mutant failed to cause fire blight disease symptoms and grew to population levels 12 orders of magnitude lower than the wild‐type. In addition, the rpoN1250::Tn5 mutant failed to elicit a hypersensitive response when infiltrated into nonhost tobacco plant leaves, and rpoN1250::Tn5 cells failed to express HrpN protein when grown in hrp (hypersensitive response and pathogenicity)‐inducing liquid medium. A plasmid‐borne copy of the wild‐type rpoN gene complemented all the rpoN1250::Tn5 mutant phenotypes tested. The rpoN1250::Tn5 mutant was prototrophic on minimal solid and liquid media, indicating that the rpoN1250::Tn5 nonpathogenic phenotype was not caused by a defect in basic metabolism or growth. This study provides clear genetic evidence that rpoN is an essential virulence gene of E. amylovora, suggesting that rpoN has the same function in E. amylovora as in P. syringae and Pe. carotovorum.