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1

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Evidence that Ralstonia eutropha (Alcaligenes eutrophus) contains a functional homologue of the Ralstonia solanacearum Phc cell density sensing system (2000)

Garg, Ram P. ; Yindeeyoungyeon, Wandee ; Gilis, Anja ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 38 (2000), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: In the phytopathogen Ralstonia (Pseudomonas) solanacearum, control of many virulence genes is partly mediated by the Phc cell density sensing system. Phc uses a novel self-produced signal molecule [3-hydroxypalmitic acid methyl ester (3-OH PAME)], an atypical two-component system (PhcS/PhcR), and a LysR-type activator (PhcA) to regulate a reversible switching between two different physiological states. While Phc is present in most R. solanacearum strains, it is apparently absent from other pseudomonad plant pathogens and prokaryotic genomes that have been sequenced. Here, we report discovery of a phcA orthologue in the non-pathogenic, facultative chemolithoautotroph Ralstonia eutropha (Alcaligenes eutrophus) that fully complements R. solanacearum phcA mutants. We also demonstrate that some R. eutropha produce an extracellular factor that complements R. solanacearum mutants deficient in production of the 3-OH PAME signal molecule that controls phcA. Additionally, Southern blot hybridization analysis suggested that R. eutropha harbours other Phc components, such as PhcB (a biosynthetic enzyme for 3-OH PAME) and PhcS (a 3-OH PAME-responsive sensor kinase). Analysis of a phcA-null mutant of R. eutropha showed that phcA (and probably Phc) positively activates motility, in contrast to R. solanacearum where it represses motility. Similarly, the R. eutropha phcA mutant was unaffected in siderophore production, whereas inactivation of phcA in R. solanacearum increases siderophore production. Although our data strongly suggest that R. eutropha has a functional Phc-like system and support the phylogeny of Ralstonia, it implies that Phc may have a different physiological and ecological function in R. eutropha.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2000.02131.x

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2

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Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum (1997)

Flavier, Albert B. ; Clough, Steven J. ; Schell, Mark A. ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 26 (1997), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Expression of virulence genes in Ralstonia solanacearum, a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs. Production of several virulence determinants is co-ordinately reduced by inactivation of phcB, but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wild-type strains of R. solanacearum. The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography. Gas chromatography and mass spectroscopy identified 3-hydroxypalmitic acid methyl ester (3-OH PAME) as the major component in the single peak of VEF activity. Authentic 3-OH PAME and the purified VEF were active at ≤1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant. Authentic 3-OH PAME also increased the production of three virulence factors by a phcB mutant over 20-fold to wild-type levels, restored normal cell density-associated expression of eps and increased expression of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small-molecule S-adenosylmethionine-dependent methyltransferase, which might catalyse synthesis of 3-OH PAME from a naturally occurring fatty acid. Biologically active concentrations of extracellular 3-OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild-type R. solanacearum. Other than acyl-homoserine lactones, 3-OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long-distance intercellular communication.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1997.5661945.x

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3

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VsrA, a second two-component sensor regulating virulence genes of Pseudomonas solanacearum (1994)

Schell, Mark A. ; Denny, Timothy P. ; Huang, Jianzhong

Oxford, UK : Blackwell Publishing Ltd

Molecular microbiology 11 (1994), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The wilt-inducing phytopathogen Pseudomonas solanacearum produces several extracellular virulence factors, both polysaccharides (EPS I) and proteins (EXPs), which are independently regulated by a LysR-type transcriptional regulator, PhcA, and a histidine kinase sensor, VsrB. Here we characterize a third locus, vsrA, which is also required for normal production of EPS I, some EXPs and wilt disease. Analysis of eps::lacZ reporters in vsrA mutants showed that, like vsrB and phcA, vsrA is required for maximal expression (transcription) of eps, which contains some of the genes necessary for production of EPS I. Unlike vsrB and phcA mutants, however, eps transcription (and EPS I production) by vsrA mutants varies from 3 to 17% of wild-type levels, depending on growth conditions. Inactivation of vsrA also causes a dramatic reduction in production of three species of EXPs (28kDa, 48kDa, and 66kDa), and an apparent increase in production of a few other EXPs. Unlike most other EPS-deficient P. solanacearum strains, vsrA mutants caused almost no disease symptoms when 104 cells were stem-inoculated into tomato plants. This correlated with a greater than 10-fold reduction in their ability to grow in plants. vsrA was cloned from a P. solanacearum genomic library by complementation of the vsrA mutant and was further subcloned on a 2.3kb DNA fragment. PhoA fusion analysis and subcellular localization of the vsrA gene product in Escherichia coli maxicells suggest that it is a 53 kDa membrane-associated protein. Analysis of the nucleotide sequence of vsrA revealed a 502 residue open reading frame with homology to the histidine kinase domain of sensors in the two-component regulator family. This discovery shows that EPS I production by P. solanacearum is simultaneously controlled by dual two-component sensors.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.1994.tb00330.x

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4

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An RpoS (σS) homologue regulates acylhomoserine lactone-dependent autoinduction in Ralstonia solanacearum (1998)

Flavier, Albert B. ; Schell, Mark A. ; Denny, Timothy P.

Oxford BSL : Blackwell Science Ltd, UK

Molecular microbiology 28 (1998), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Many bacteria sense an appropriate growth condition or a critical population density for gene expression by producing acylhomoserine lactones (acyl-HSLs) that act as intercellular autoinduction signals. We recently showed that, in Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium, acyl-HSL production requires solI, which encodes a putative acyl-HSL synthase, and that its expression is positively regulated by the acyl-HSL-responsive SolR transcriptional regulator. This acyl-HSL-dependent autoinduction system is noteworthy because (i) it is regulated by a ‘higher level’ autoinducer system (responsive to 3-hydroxypalmitic acid methyl ester) via PhcA, a LysR-type transcriptional regulator and (ii) acyl-HSL production requires two additional unlinked loci. As reported here, cloning and sequencing of one of these other loci revealed that it encodes a homologue of RpoS, an alternative sigma factor (σS) that in other bacteria activates gene expression during stationary phase or in response to stress conditions. R. solanacearum RpoS (RpoSRso) was demonstrated to function as a σ factor because when introduced in trans into an Escherichia coli rpoS mutant it largely restored expression of the RpoS-dependent bolAp1 gene. Mutation of rpoSRso in R. solanacearum reduced survival during starvation and low pH conditions, but did not affect survival during exposure to hydrogen peroxide, high osmolarity or high temperature. This mutant was also altered in its production of several virulence factors and wilted tomato plants several days more slowly than the wild-type parent. Transcription of solR and solI were decreased in an rpoSRso background (thereby reducing acyl-HSL production), but neither mutations in solR, solI or phcA nor addition of acyl-HSLs affected rpoSRso expression. Therefore, in R. solanacearum the acyl-HSL-dependent autoinduction system is controlled both by a second autoinduction system and by the RpoSRso sigma factor.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1998.00804.x

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5

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Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence (2002)

Kang, Yaowei ; Liu, Huanli ; Genin, Stéphane ; [et al.]

Oxford, UK : Blackwell Science, Ltd

Molecular microbiology 46 (2002), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: As reported previously for Ralstonia solanacearum strain GMI1000, wild-type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching-positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43-amino-acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10-fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10-fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2002.03187.x

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6

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Autoregulator-dependent Control of Extracellular Polysaccharide Production in Phytopathogenic Bacteria (1999)

Denny, Timothy P.

Springer

European journal of plant pathology 105 (1999), S. 417-430

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ISSN: 1573-8469

Keywords: autoinduction ; Pantoea stewartii ; pheromone ; quorum sensing ; Ralstonia solanacearum ; Xanthomonas campestris

Source: Springer Online Journal Archives 1860-2000

Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition

Notes: Abstract Extracellular polysaccharides (EPSs) likely provide phytopathogenic bacteria a selective advantage both inside and outside plants. Despite the relatively scant knowledge about EPS biosynthesis in phytopathogenic bacteria, it clearly is a well controlled, complex, energy-intensive process. Unexpectedly, three phytopathogenic bacteria have been found to autoregulate EPS production in response to extracellular signal compounds (pheromones) that they produce. Like many bacteria, Pantoea stewartii subsp. stewartii produces a N-acyl-homoserine lactone (AHL) autoinducer. However, unlike most AHL-dependent autoinduction systems, that in P. stewartii subsp. stewartii somehow represses EPS production in the absence of autoinducer. Instead of an AHL-dependent system (which it also has), Ralstonia solanacearum uses a novel autoregulator identified as 3-hydroxypalmitic acid methyl ester to regulate EPS biosynthesis. A lack of this autoregulator in R. solanacearum results in repression of EPS biosynthesis by a complex two-component sensor/response regulator signal cascade. Xanthomonas campestris pv. campestris has two partially overlapping autoregulatory systems. The autoregulators are incompletely characterized, but one diffusible signal factor (DSF) is thought to be a fatty acid derivative and the other diffusible factor (DF) may be a butyrolactone. The autoregulation pathways in X. campestris pv. campestris are essentially unknown, but EPS production is controlled by both the DSF and DF systems, whereas production of extracellular enzymes and pigment production are regulated independently. In a confined micro-environment, population density and intercellular concentrations of an autoregulator will increase in parallel, so autoregulation is one way that bacteria can coordinate gene expression to synthesize EPS only at high cell density. However, because there is often limited evidence that it is actually cell density that is being detected, researchers should not assume a priori that autoregulation must function for quorum sensing. Some possible reasons for why phytopathogenic bacteria would benefit from delaying EPS production are discussed.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1008767931666

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